i 

 

 

 

 

 

THE VIRAL AUTHORS OF EVOLUTION 

FROM SEQUENCE TO STRUCTURE 

by 

 

William Stevens Henriques 

 

 

 

 

 

 

A dissertation submitted in partial fulfillment 

of the requirements for the degree 

 

of 

 

Doctor of Philosophy 

 

in 

 

Microbiology and Immunology 

 

 

 

MONTANA STATE UNIVERSITY 

Bozeman, Montana 

 

 

 

December 2024



 

 

 

 

 

 

 

 

©COPYRIGHT 

by 

William Stevens Henriques 

2024 

All Rights Reserved



ii 

 

 

DEDICATION 

This dissertation is dedicated to E. coli and elephants, and all the biology in between. 

  



iii 

 

 

ACKNOWLEDGEMENTS 

Science is an adventure into the unknown that requires curiosity, boldness, and luck. But 

science is also coming back with a new kernel of knowledge, which requires grit, creativity, and 

clarity. I first want to thank my mentor Blake Wiedenheft for teaching me these lessons. Working 

with Blake has made me a better storyteller and scientist. Science is also filled with failures and 

missteps. I thank my co-mentor Harmit Malik, who has reminded me that failures and missteps 

are part of the process, and the most important thing is to return to the lab day in and day out 

with both compassion and enthusiasm.  

No one ventures into the unknowns of science alone. Mentors and colleagues offer a 

helping hand in the squalls of failures, tedium, and rejection. I would like to thank Anna, Artem, 

Andrew, Royce, and Janet for their mentorship, as well as Murat, Tanner, and Calvin for their 

bioinformatic companionship and Nate for many shared hours at the microscope. A special 

thanks to Dr. Martin Lawrence and Colin Gauvin at Montana State’s cryoEM Facility, the 

Research Cyberinfrastructure team and especially Coltran Hophan-Nichols, and my graduate 

committee for their encouragement and guidance.  

Finally, I want to acknowledge my friends who kept me sane and singing, the wonderful 

crew at Blackbird kitchen who kept me connected to the Bozeman community, and my family, 

who was always on the other end of the phone during those long dark nights on Everest.  

This work was funded in part by the National Institutes of Health (R35GM134867), the 

M.J. Murdock Charitable Trust, a young investigator award to Blake Wiedenheft from Amgen, 

and the Montana State University Agricultural Experimental Station (USDA NIFA). 

  



iv 

 

 

TABLE OF CONTENTS 

1. INTRODUCTION .............................................................................................................. 1 

Conserved Viral Defense Systems ...................................................................................... 2 
Unique Viral Defense Systems ........................................................................................... 2 
Abundance Leads to Co-option For Defense ...................................................................... 4 
Domestication Beyond Defense .......................................................................................... 5 
Sequence and Structure ....................................................................................................... 6 
Dissertation Overview ........................................................................................................ 7 
References ......................................................................................................................... 12 

2. GENOME SEQUENCE, PHYLOGENETIC ANALYSIS, AND 

STRUCTURE BASED ANNOTATION REVEALS METABOLIC 

POTENTIAL OF CHLORELLA SP. SLA-04 ................................................................... 20 

Contribution of Authors and Co-Authors ......................................................................... 20 
Manuscript Information .................................................................................................... 22 
Abstract ............................................................................................................................. 23 
Introduction ....................................................................................................................... 24 
Methods............................................................................................................................. 25 

Strain Information ................................................................................................. 25 
Strain Cultivation Conditions ................................................................... 25 
Axenic Isolation ........................................................................................ 26 

Genome Sequencing and Assembly ...................................................................... 26 
High Molecular Weight DNA Extraction .................................................. 26 
RNA Extraction ......................................................................................... 27 
DNA and RNA Sequencing ...................................................................... 28 
Genome Assembly and Annotation ........................................................... 28 

Comparative Analyses .......................................................................................... 30 
Phylogenetic Analysis. .............................................................................. 30 

Results ............................................................................................................................... 31 
General Genome Characteristics ........................................................................... 31 

Chlorella sp. SLA-04 Genome Assembly Statistics ................................. 31 
Genome Completeness Analysis ............................................................... 31 
Annotation of Chlorella Sp. SLA-04 ........................................................ 32 
Annotation Using Protein 3D Structural Homology ................................. 33 
Phylogenetic Analysis of Chlorella Sp. SLA-04....................................... 35 

Genomic Analysis of Metabolic Pathways in Chlorella Sp. SLA-04 ................... 35 
Discussion ......................................................................................................................... 37 
Conclusion ........................................................................................................................ 40 
Data Access ....................................................................................................................... 40 
Acknowledgements ........................................................................................................... 40 
References ......................................................................................................................... 40 



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TABLE OF CONTENTS CONTINUED 

Supplementary Figures ..................................................................................................... 48 
Supplementary Tables ....................................................................................................... 52 

3. CLARIFYING CRISPR: WHY REPEATS IDENTIFIED IN THE HUMAN 

GENOME SHOULD NOT BE CONSIDERED CRISPRS .............................................. 54 

Contribution of Authors and Co-Authors ......................................................................... 54 
Manuscript Information .................................................................................................... 55 
Abstract ............................................................................................................................. 56 
Introduction ....................................................................................................................... 56 
Methods............................................................................................................................. 57 

CRISPR Predictions .............................................................................................. 57 
Identification of Cas Genes in the Human Genome ............................................. 59 
Comparison of Repeat Length Distribution of CRISPRs from 

Prokaryotes and HCRISPRs ................................................................................. 59 
Results and Discussion ..................................................................................................... 60 
References ......................................................................................................................... 65 
Supplementary Figures ..................................................................................................... 68 
Supplemental Information ................................................................................................ 69 

4. THE DIVERSE EVOLUTIONARY HISTORIES OF DOMESTICATED 

METAVIRAL CAPSID GENES IN MAMMALS ........................................................... 73 

Contribution of Authors and Co-Authors ......................................................................... 73 
Manuscript Information .................................................................................................... 74 
Abstract ............................................................................................................................. 75 
Introduction ....................................................................................................................... 76 
Results ............................................................................................................................... 80 

Divergent Metaviral Capsid-derived Genes in the Human Genome .................... 80 
Domesticated Metaviral Capsid Genes Experienced Distinct 

Evolutionary Retention Fates ................................................................................ 84 
Domesticated Metaviral Capsid Genes are Retained under 

Purifying and Positive Selection ........................................................................... 89 
Domesticated Capsid Genes are Structurally Diverse .......................................... 93 
Diverse N-terminal Structures in Domesticated Genes Derived 

from Metaviral Ancestors ..................................................................................... 98 
A Novel Putative RNA-binding Domain in the PNMA Family ......................... 101 

Discussion ....................................................................................................................... 105 
Methods............................................................................................................................ 111 

Building Capsid HMMs ....................................................................................... 111 
Identifying Capsid Genes in Vertebrate Genomes ...............................................113 

 



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TABLE OF CONTENTS CONTINUED 

Matching Human Capsid-like ORF Sequences to Previously 

Annotated Genes ..................................................................................................114 
Analysis of Domain Architecture and Genetic Context .......................................114 
Phylogenetic Analysis of Capsid-like Sequences ................................................114 
Identification of Mammalian Orthologs ..............................................................115 
Analysis of Evolutionary Selective Constraints ..................................................119 
Structure Predictions and Homology Modelling ................................................ 121 
Phylogenetic Analysis of Putative RBD Sequences ........................................... 122 
Expression Analysis ............................................................................................ 123 
International Mouse Phenotyping Consortium ................................................... 123 

Data and Resource Availability ....................................................................................... 123 
References ....................................................................................................................... 124 
Supplementary Figures ................................................................................................... 136 
Supplementary Tables ..................................................................................................... 153 

5. THE RETROTRANSPOSON-DERIVED CAPSID GENES PNMA1 AND 

PNMA4 MAINTAIN REPRODUCTIVE CAPACITY ................................................... 159 

Contribution of Authors and Co-Authors ....................................................................... 159 
Manuscript Information .................................................................................................. 162 
Abstract ........................................................................................................................... 163 
Introduction ..................................................................................................................... 163 
Results ............................................................................................................................. 165 

Expression of PNMA1 and PNMA4 Declines with Age in Human 

Ovaries ................................................................................................................ 165 
Male and Female Mice Lacking Pnma1 or Pnma4 Show Premature 

Loss of Fertility ................................................................................................... 168 
Pnma1 and Pnma4 Mutants Acquire Abdominal Obesity but 

Appear Behaviorally Normal .............................................................................. 173 
PNMA1 and PNMA4 Form Capsid-like Structures in Gonadal 

Tissue .................................................................................................................. 175 
Human Variation at the PNMA1 and PNMA4 Loci is Associated 

with Reproductive Defects .................................................................................. 178 
Discussion ....................................................................................................................... 179 
Methods........................................................................................................................... 183 

Generation of Mutant Mouse Models ................................................................. 183 
Mouse Husbandry ............................................................................................... 184 
Mouse Genotyping .............................................................................................. 185 
Fertility Assay ..................................................................................................... 186 
Body and Gonad Weight ..................................................................................... 186 
Immunofluorescence of Testis Sections .............................................................. 186 
Epididymal Sperm Counts .................................................................................. 188 



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TABLE OF CONTENTS CONTINUED 

Periodic Acid-Schiff (PAS) Staining ................................................................... 188 
Blood Collection, Sample Preparation, and Hormone Analysis ......................... 189 
Ovarian Histology ............................................................................................... 189 
Oocyte Collection and Maturation ...................................................................... 189 
Mouse Oocyte Isolation, Culturing, Microinjection and High-

Resolution Confocal Microscopy ....................................................................... 190 
Plasmid Creation ................................................................................................. 191 
HEK293T Cell Culture ....................................................................................... 191 
HEK293T Transfection ....................................................................................... 192 
Cloning, Expression, and Purification of Recombinant PNMA1 

and PNMA4 ........................................................................................................ 193 
Negative Stain Transmission Electron Microscopy ............................................ 194 
Velocity Gradient Ultracentrifugation................................................................. 195 
Western Blotting ................................................................................................. 195 
Quantitative PCR ................................................................................................ 197 
Extracellular Protein Analysis ............................................................................. 198 
Analysis of Testicular Capsid-Like PNMA4 Protein .......................................... 198 

Lysate Preparation ................................................................................... 198 
Double Sucrose Gradient ........................................................................ 199 
Iodixanol Step Gradient .......................................................................... 199 

Northern Blot Analysis ....................................................................................... 199 
PNMA1-5 Expression Analysis in Human and Mouse Ovary ............................ 200 
Analysis of Traits Associated with Human PNMA1 and PNMA4 

Variation .............................................................................................................. 201 
Statistical Analysis .............................................................................................. 201 
Neurobehavioral Tests ......................................................................................... 201 

Open Field Test ....................................................................................... 202 
Grip Strength Test ................................................................................... 202 
Inverted Hang Test .................................................................................. 202 
Y Maze Spontaneous Alternation ........................................................... 203 
Fear Conditioning ................................................................................... 203 

References ....................................................................................................................... 204 
Supplementary Figures ................................................................................................... 212 
Supplementary Tables ..................................................................................................... 224 

6. A VIRALLY-ENCODED TRNA NEUTRALIZES THE PARIS 

ANTIVIRAL DEFENSE SYSTEM................................................................................ 228 

Contribution of Authors and Co-Authors ....................................................................... 228 
Manuscript Information .................................................................................................. 231 
Abstract ........................................................................................................................... 232 
Introduction ..................................................................................................................... 232 



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TABLE OF CONTENTS CONTINUED 

Results ............................................................................................................................. 234 
PARIS Forms a Propeller-shaped Complex ........................................................ 234 
AriA Sequesters AriB .......................................................................................... 236 
AriA Binds to Ocr and Releases AriB................................................................. 239 
Activated PARIS Inhibits Translation ................................................................. 244 
Triggers and a Suppressor of PARIS .................................................................. 245 
Viral TRNA Resistant to AriB Cleavage............................................................. 246 
Evolution of the PARIS Defense System ............................................................ 249 

Discussion ....................................................................................................................... 250 
Methods........................................................................................................................... 252 

Bacterial Strains, Phages, and Plasmids ............................................................. 252 
Expression and Purification of the PARIS Complex .......................................... 253 
In Vivo Protein Pull-down ................................................................................... 254 
Glutaraldehyde Cross-linking Assay................................................................... 255 
In Vitro Protein Pull-down .................................................................................. 255 
Cryo-EM Sample Preparation and Data Collection ............................................ 256 
Cryo-EM Data Processing of Inactive PARIS .................................................... 257 
Cryo-EM Data Processing of Ocr Pulldown ....................................................... 258 
Model Building and Refinement ......................................................................... 258 
ATPase Assays .................................................................................................... 259 
Efficiency of Plaquing Assay .............................................................................. 260 
Liquid Culture Phage Infection ........................................................................... 261 
Solid Media Toxicity Assay ................................................................................ 261 
Liquid Culture Toxicity Assay ............................................................................ 262 
Fluorescence Microscopy ................................................................................... 262 
Extraction of Total DNA ..................................................................................... 263 
TUNEL Assay ..................................................................................................... 263 
Hi-C Procedure and Sequencing ......................................................................... 264 
Metabolic Labeling ............................................................................................. 265 
In Vitro Translation ............................................................................................. 266 
Isolation and Sequencing of T5 Escaper Mutants ............................................... 267 
Extraction of Total RNA ..................................................................................... 268 
Preparation of Enriched Small RNA Fractions from the Total RNA 

Samples ............................................................................................................... 269 
Cleavage Assay by AriB ..................................................................................... 270 
Mapping of the AriB Cleavage Site by Primer Extension .................................. 270 
Mapping the AriB Cleavage Site by Specific Reverse Transcription 

(RT) and Sanger Sequencing .............................................................................. 271 
Northern Blot ...................................................................................................... 272 
Defense System Detection .................................................................................. 272 
Domain Annotation ............................................................................................. 273 



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TABLE OF CONTENTS CONTINUED 

AriA and AriB Tree ............................................................................................. 273 
Defense System ATPase AAA15/21 Tree ........................................................... 274 
Statistics and Reproducibility ............................................................................. 274 

References ....................................................................................................................... 274 
Extended Data Figures .................................................................................................... 280 
Extended Data Tables ...................................................................................................... 291 
Supplementary Tables ..................................................................................................... 292 

7. STRUCTURE REVEALS WHY GENOME FOLDING IS NECESSARY 

FOR SITE-SPECIFIC INTEGRATION OF FOREIGN DNA INTO 

CRISPR ARRAYS .......................................................................................................... 312 

Contribution of Authors and Co-Authors ....................................................................... 312 
Manuscript Information .................................................................................................. 314 
Abstract ........................................................................................................................... 315 
Introduction ..................................................................................................................... 315 
Results ............................................................................................................................. 318 

Cryo-EM Structure of Type I-F CRISPR Integration Complex ......................... 318 
Foreign DNA Constrains Cas2/3 Linker against Cas1 ....................................... 322 
Cas2 Homodimers Recognize and Bend Inverted Repeat 

Sequences ............................................................................................................ 324 
Cas2 Homodimer is Surrounded by Four DNA Helices ..................................... 326 
IHF and the Leader Direct Integration into Diverse Sequences ......................... 326 
5’ GT is Critical for Cas1-mediated Integration ................................................. 329 

Discussion ....................................................................................................................... 330 
Methods........................................................................................................................... 335 

Nucleic Acid Preparation .................................................................................... 335 
Cas1 and Cas2/3 Mutagenesis ............................................................................ 336 
Protein Purification ............................................................................................. 336 
In Vitro Integration Assays .................................................................................. 338 
Assembly and Purification of I-F Integration Complex ..................................... 339 
Cryo-EM Sample Preparation and Data Acquisition .......................................... 340 
Cryo-EM Image Processing ................................................................................ 342 
Model Building and Validation ........................................................................... 343 
Cas1, Cas2/3, and Repeat Conservation Analysis .............................................. 344 
Data Availability ................................................................................................. 347 

Extended Data Figures .................................................................................................... 347 
References ....................................................................................................................... 358 
Supplementary Tables ..................................................................................................... 366 

 

 



x 

 

 

TABLE OF CONTENTS CONTINUED 

8. STRUCTURES REVEAL HOW A CRISPR INTEGRASE CAPTURES, 

DELIVERS, AND INTEGRATES FOREIGN DNA INTO THE CRISPR 

ARRAY ........................................................................................................................... 374 

Contribution of Authors and Co-Authors ....................................................................... 374 
Manuscript Information .................................................................................................. 375 
Abstract ........................................................................................................................... 376 
Introduction ..................................................................................................................... 376 
Results ............................................................................................................................. 378 

Cas1-2/3 Forms a DNA Capture Complex ......................................................... 378 
Structural Basis for Cas3 HD Nuclease and RecA Helicase 

Inhibition ............................................................................................................. 382 
Cas3 Blocks Foreign DNA Integration ............................................................... 385 
Foreign DNA Capture Exposes Genome Binding Sites ..................................... 386 
Structure Precludes a Role for Cas3 in PAM Trimming ..................................... 390 
Foreign DNA Binding Exacerbates Asymmetric Instability ............................... 391 
The PAM Changes the Stability of the Integration Complex ............................. 391 
The CRISPR Repeat Channel Stretches and Distorts The Repeat ...................... 395 

Discussion ....................................................................................................................... 396 
Materials & Methods ...................................................................................................... 399 

Conservation Analysis of Cas2/3 and Cas1 ........................................................ 399 
Nucleic Acid Preparation .................................................................................... 400 
Protein Purification ............................................................................................. 400 
Assembly and Purification of the Delivery Complex ......................................... 402 
Assembly and Purification of the Integration Complex ..................................... 402 
CryoEM Sample Preparation and Data Acquisition – Cas1-2/3 

Capture Complex ................................................................................................ 403 
CryoEM Sample Preparation and Data Acquisition – Cas1-2/3 

Delivery Complex ............................................................................................... 403 
CryoEM Sample Preparation and Data Acquisition – Cas1-2/3 

Integration Complex ........................................................................................... 404 
Cryo-EM Image Processing ................................................................................ 405 
Model Building and Validation ........................................................................... 405 

References ....................................................................................................................... 406 
Supplementary Figures ................................................................................................... 414 
Supplementary Tables ..................................................................................................... 424 

9. CONCLUSION ............................................................................................................... 426 

From Sequence to Structure ............................................................................................ 428 
Structure Determines Function ....................................................................................... 429 
Concluding Remarks ....................................................................................................... 430 



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TABLE OF CONTENTS CONTINUED 

References ....................................................................................................................... 431 

CUMULATIVE REFERENCES CITED .............................................................................. 433 

APPENDIX: RAPID CRYOEM STRUCTURE DETERMINATION FROM 

CELL-FREE TRANSCRIPTION AND TRANSLATION ................................................... 493 

Abstract ........................................................................................................................... 494 
Main Text ........................................................................................................................ 494 
References ....................................................................................................................... 498 

  



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LIST OF TABLES 

Table                Page 

1. Supplementary Table 2.1. Genome statistics of Chlorella sp. SLA-04 

genome. ............................................................................................................................. 52 

2. Supplementary Table 2.2. Summary of repetitive elements identified by 

RepeatMasker using Dfam and Repbase as a combined reference database. ................... 53 

3. Supplementary Table 2.3. Active transcription of lipid synthesis genes 

within the Chlorella sp. SLA-04 genome. ........................................................................ 53 

4. Supplementary Table 4.1. Model statistics for custom capsid Hidden 

Markov Models (HMMs) build from multiple sequence alignments (MSA) 

of capsid amino acid sequences. ..................................................................................... 153 

5. Supplementary Table 4.2. Gene names table. ................................................................. 155 

6. Supplementary Table 4.3. Table of genomes used in this study ..................................... 156 

7. Supplementary Table 4.4. BLAST statistics for consensus Repbase 

metaviral sequences used as proxies for ancestral metaviruses ...................................... 157 

8. Supplementary Table 4.5. Phenotypes associated with knockouts of 

domesticated metaviral genes in mice. ........................................................................... 158 

9. Supplementary Table 5.1. Traits associated with PNMA1 and PNMA4 

variants in the human population. ................................................................................... 227 

10. Extended Data Table 6.1. Cryo-EM data collection, processing, and model 

validation......................................................................................................................... 291 

11. Supplementary Table 1. Bacterial strains, phages, and plasmids used in the 

study ................................................................................................................................ 299 

12. Supplementary Table 2. Primers used for cloning ...........................................................311 

13. Supplementary Table 3. Nucleotide sequence of the probes used for 

Northern blot and in primer extension .............................................................................311 

14. Table 7.1. Cryo-EM data collection, refinement, and validation statistics ..................... 321 

15. Supplementary Table 1. Oligonucleotides used in this study. ......................................... 373 

16. Supplementary Table 8.1. Refinement statistics for the Cas1-2/3 complex ................... 424 



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LIST OF TABLES CONTINUED 

17. Supplementary Table 8.2. Refinement statistics for Cas1-2/3 bound to 

dsDNA fragment ............................................................................................................. 425 

 

  



xiv 

 

 

LIST OF FIGURES 

Figure                Page 

1. Figure 2.1. Complete genome of Chlorella sp. SLA-04. .................................................. 32 

2. Figure 2.2. Annotation using protein structural similarity improves 

functional understanding of SLA-04 proteome. ............................................................... 33 

3. Figure 2.3. Phylogenomic analysis of green algae performed using 

orthologous genes indicates that SLA-04 is distinct from other Chlorella 

sorokiniana species. .......................................................................................................... 34 

4. Figure 2.4. Flow of carbon from fixation to lipid synthesis in Chlorella sp. 

SLA-04. ............................................................................................................................. 36 

5. Supplementary Figure 2.1. DNA isolation and sequencing parameters for 

Chlorella sp. SLA-04. ....................................................................................................... 48 

6. Supplementary Figure 2.2. Phylogenetic tree of LTR retrotransposon capsid 

domains places SLA-04 retrotransposons among Copia/Pseudoviridae LTR 

retrotransposons. ............................................................................................................... 49 

7. Supplementary Figure 2.3. Phylogenomic analysis of green algae performed 

using 18S rDNA. ............................................................................................................... 50 

8. Supplementary Figure 2.4. Presence of ATP citrate lyase and sphingolipid 

enzymes across algal genomes.......................................................................................... 51 

9. Figure 3.1. Comparison of architectural features in CRISPRs and 

hCRISPRs. ........................................................................................................................ 60 

10. Figure 3.2. Repetitive elements in the human genome do not contain 

architectural features consistent with CRISPR loci .......................................................... 62 

11. Supplementary Figure 3.1. Previously reported CRISPR-like elements in 

the mitochondrial genome of a plant (Vicia faba) and the genome of a giant 

mimivirus (APMV) ........................................................................................................... 68 

12. Figure 4.1. Full-length capsid-like ORFs in the human genome ...................................... 82 

13. Figure 4.2. Metaviral-derived capsid genes show distinct evolutionary 

trajectories across placental mammals. ............................................................................. 85 

 



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LIST OF FIGURES CONTINUED 

14. Figure 4.3. RTL3 and RTL10 exhibit domain-specific and lineage-specific 

patterns of conservation. ................................................................................................... 87 

15. Figure 4.4. Evolutionary rates of domesticated metaviral genes across 

placental mammals and primates. ..................................................................................... 91 

16. Figure 4.5. Four independent metavirus domestication events include 

structurally distinct N-terminal domains. ......................................................................... 94 

17. Figure 4.6. Predicted RNA-binding domain in the PNMA family and related 

metaviruses ..................................................................................................................... 102 

18. Supplementary Figure 4.1. Constructing Hidden Markov Models for the 

capsid domain of LTR retrotransposons ......................................................................... 136 

19. Supplementary Figure 4.2. Phylogeny of ARC proteins ................................................ 137 

20. Supplementary Figure 4.3. Phylogeny of the putative RNA-binding domain 

of PNMA proteins. .......................................................................................................... 139 

21. Supplementary Figure 4.4. Phylogeny of PNMA protein capsid domains. .................... 140 

22. Supplementary Figure 4.5. PNMA6E/F has duplicated multiple times in 

distinct lineages. .............................................................................................................. 142 

23. Supplementary Figure 4.6. Phylogenetic tree for SIRH/RTL family (half 

capsid alignment). ........................................................................................................... 144 

24. Supplementary Figure 4.7. Phylogenetic tree for SIRH/RTL family (full 

capsid alignment). ........................................................................................................... 145 

25. Supplementary Figure 4.8. Conservation and divergence of the capsid 

domain in the SIRH/RTL family. .................................................................................... 146 

26. Supplementary Figure 4.9. Lack of structural conservation in the N-terminal 

domain of the SIRH/RTL family. .................................................................................... 148 

27. Supplementary Figure 4.10. The evolutionary origins of the putative RNA-

binding domain of the PNMA family. ............................................................................. 150 

28. Supplementary Figure 4.11. Human expression of domesticated metaviral 

capsid genes. ................................................................................................................... 152 



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LIST OF FIGURES CONTINUED 

29. Figure 5.1. PNMA1 and PNMA4 are expressed in human ovaries. ............................... 166 

30. Figure 5.2. PNMA1 and PNMA4 are conserved among eutherians ............................... 168 

31. Figure 5.3. Male mice lacking Pnma1 or Pnma4 prematurely lose 

reproductive capacity ...................................................................................................... 170 

32. Figure 5.4. Female mice lacking Pnma1 or Pnma4 have age-dependent 

reproductive defects. ....................................................................................................... 172 

33. Figure 5.5. Pnma1 and Pnma4 mutants gain abdominal fat with age. ............................ 174 

34. Figure 5.6. PNMA proteins form capsid-like structures that can exit human 

cells ................................................................................................................................. 176 

35. Figure 5.7. Model for PNMA1 and PNMA4 function. ................................................... 179 

36. Supplementary Figure 5.1. PNMA2, PNMA3, and PNMA5 expression 

analysis in human ovaries. .............................................................................................. 212 

37. Supplementary Figure 5.2. DAZL has the capacity to post-transcriptionally 

regulate PNMA1 and PNMA4. ....................................................................................... 213 

38. Supplementary Figure 5.3. Diagram of Pnma1 and Pnma4 loci on M. 

musculus chromosome 12 ............................................................................................... 214 

39. Supplementary Figure 5.4. Defective testicular and ovarian morphology in 

Pnma1 and Pnma4 mutants ............................................................................................. 216 

40. Supplementary Figure 5.5. Analysis of undifferentiated spermatogonial 

cells by PLZF staining .................................................................................................... 217 

41. Supplementary Figure 5.6. Several ovarian features are not dramatically 

altered in Pnma1 and Pnma4 mutants. ............................................................................ 218 

42. Supplementary Figure 5.7. Oocytes that enter meiosis are largely unaffected 

in Pnma1 and Pnma4 mutants. ........................................................................................ 219 

43. Supplementary Figure 5.8. Single-cell analysis of Pnma1 and Pnma4 

expression in aging mouse ovaries ................................................................................. 220 

44. Supplementary Figure 5.9. Pnma1 and Pnma4 are expressed in 

subpopulations of ovarian cells ....................................................................................... 221 



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LIST OF FIGURES CONTINUED 

45. Supplementary Figure 5.10. Mice lacking Pnma1 or Pnma4 exhibit normal 

neurobehavioral and muscular traits. .............................................................................. 222 

46. Supplementary Figure 5.11. Size exclusion chromatography of recombinant 

PNMA1 and PNMA4. ..................................................................................................... 223 

47. Supplementary Figure 5.12. PNMA1 and PNMA4 expressed in human cells 

forms capsid-sized particles. ........................................................................................... 224 

48. Figure 6.1. PARIS is a two-component system that assembles into a 

propeller-shaped supramolecular complex. .................................................................... 235 

49. Figure 6.2. Interaction between the AriA ATPase and AriB essential for 

PARIS-mediated defense. ............................................................................................... 237 

50. Figure 6.3. AriA interacts with Ocr in an ATPase-dependent manner, leading 

to the release of AriB from the PARIS complex ............................................................. 240 

51. Figure 6.4. The activation of PARIS leads to cell death and inhibition of 

translation ........................................................................................................................ 242 

52. Figure 6.5. PARIS cleaves E. coli tRNALys and T5 encodes a non-cleavable 

tRNALys. ........................................................................................................................ 247 

53. Figure 6.6. Phylogenies of PARIS and related ABC ATPase powered 

defense systems ............................................................................................................... 249 

54. Extended Data Figure 6.1. AlphaFold2 structural predictions of AriA and 

AriB................................................................................................................................. 280 

55. Extended Data Figure 6.2. Structural comparison of PARIS homologs. ........................ 281 

56. Extended Data Figure 6.3. Workflow for structural determination of the 

PARIS complex ............................................................................................................... 282 

57. Extended Data Figure 6.4. AriB toxicity is dependent on association with 

AriA ................................................................................................................................ 283 

58. Extended Data Figure 6.5. Purification of activated AriB. ............................................. 284 

59. Extended Data Figure 6.6. Structure determination of AriA purified using a 

Ocr-Strep pulldown. ........................................................................................................ 285 



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LIST OF FIGURES CONTINUED 

60. Extended Data Figure 6.7. AriA ATPase activity. ........................................................... 286 

61. Extended Data Figure 6.8. PARIS activation does not result in total RNA or 

DNA degradation ............................................................................................................ 287 

62. Extended Data Figure 6.9. PARIS activation results in DNA compactization 

akin to inhibition of translation with chloramphenicol. .................................................. 288 

63. Extended Data Figure 6.10. Identification of the T5 genomic region 

required for infection of PARIS+ cells. .......................................................................... 289 

64. Extended Data Figure 6.11. tRNALys cleavage product and phylogenetics 

of PARIS ......................................................................................................................... 290 

65. Figure 7.1. Cryo-EM structure of the type I-F CRISPR integration complex

......................................................................................................................................... 319 

66. Figure 7.2. Foreign DNA constrains the Cas2/3 linker against conserved 

Cas1 residues. ................................................................................................................. 323 

67. Figure 7.3. The Cas2 homodimer simultaneously coordinates four dsDNA 

helices critical to CRISPR integration. ........................................................................... 325 

68. Figure 7.4. Sequence motifs in the leader and IHF proteins facilitate Cas1-

2/3-based integration into diverse repeat sequences. ...................................................... 328 

69. Figure 7.5. I-F CRISPR integration complex suggests a mechanism for 

primed acquisition and Cascade impact on integration. ................................................. 331 

70. Figure 7.6. DNA is a flexible scaffold that controls DNA mobilization. ........................ 333 

71. Extended Data Figure 7.1. Cryo-EM sample preparation, imaging and 

processing for type I-F integration complex ................................................................... 349 

72. Extended Data Figure 7.2. Cas1-2/3 undergoes a large structural 

rearrangement during integration. ................................................................................... 350 

73. Extended Data Figure 7.3. Conservation analysis of Cas1-2/3 residues 

involved in DNA binding and integration. ...................................................................... 351 

74. Extended Data Figure 7.4. Cas1-2/3 predominantly recognizes IR motifs, 

CRISPR repeat and foreign DNA through non-sequence specific 

interactions. ..................................................................................................................... 352 



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LIST OF FIGURES CONTINUED 

75. Extended Data Figure 7.5. Purification of structure-guided mutants of Cas1-

2/3 ................................................................................................................................... 353 

76. Extended Data Figure 7.6. Validation of Cas1-2/3 interactions with the 

foreign DNA and IR motifs............................................................................................. 354 

77. Extended Data Figure 7.7. PAM blocks Cas-mediated integration of foreign 

DNA into CRISPR repeat. .............................................................................................. 355 

78. Extended Data Figure 7.8. Control reactions for integration assay and 

generation of 32P-labelled ladder. .................................................................................. 356 

79. Extended Data Figure 7.9. Validation of Cas1-2/3 interactions with the 

repeat. .............................................................................................................................. 357 

80. Figure 8.1. One side of the Cas1-2/3 integrase has a positively charged 

foreign DNA-binding channel. ....................................................................................... 379 

81. Figure 8.2. The Cas3 active sites are blocked in the DNA capture complex. ................. 384 

82. Figure 8.3. Foreign DNA triggers a conformational rearrangement that 

exposes leader recognition sites and preserves Cas3 inhibition ..................................... 387 

83. Figure 8.4. The PAM blocks the second transesterification by altering 

complex stability. ............................................................................................................ 392 

84. Figure 8.5. The repeat channel distorts the CRISPR repeat............................................ 395 

85. Supplementary Figure 8.1. Purification and data processing of the Cas1-2/3 

capture complex. ............................................................................................................. 414 

86. Supplementary Figure 8.2 Cas1 in complex with Cas2/3 preserves dimeric 

architecture. ..................................................................................................................... 415 

87. Supplementary Figure 8.3. Conservation of the four-bladed propeller Cas1-

2/3 architecture across type I-F systems ......................................................................... 416 

88. Supplementary Figure 8.4. The Cas2 C-terminus determines the extent of 

complex interactions ....................................................................................................... 417 

89. Supplementary Figure 8.5. Positively charged foreign DNA binding 

residues are conserved in the Cas1-2/3 complex ............................................................ 418 



xx 

 

 

LIST OF FIGURES CONTINUED 

90. Supplementary Figure 8.6. A conserved domain inserted into the HD active 

site explains nuclease inhibition ..................................................................................... 419 

91. Supplementary Figure 8.7. Three interfaces clamp Cas3 in place, inhibiting 

both interference and integration interactions ................................................................. 420 

92. Supplementary Figure 8.8. Purification and data processing of the Cas1-2/3 

capture complex .............................................................................................................. 421 

93. Supplementary Figure 8.9. Purification and cryoEM data analysis of PAM-

containing integration complex....................................................................................... 422 

94. Supplementary Figure 8.10. Positively charged residues in the repeat 

channel are conserved in type I-F CRISPR systems. ...................................................... 423 

95. Figure A.1. Sequence to structure in 24 hours ................................................................ 495 

96. Figure A.2.  TxTl cryoEM produces high resolution structures ..................................... 497 

  



xxi 

 

 

ABSTRACT 

 Viruses are the most abundant biological entities on the planet. Like any parasite, these 

genetic parasites require a host. Inside a host cell, viruses hijack the transcription and translation 

machinery to replicate. In response to the ubiquitous threat of viral predation, host cells have 

evolved an astonishing diversity of mechanisms to stop, control, and even repurpose pieces of 

viruses. In this dissertation, I use bioinformatic, biochemical, and structural approaches to show 

this ancient conflict shapes host evolution by constantly rewriting the host genome. Research 

conducted during this dissertation has spanned the domains of life with three major foci. First, I 

have used bioinformatics to identify genetic parasites and defense systems embedded within 

algal and human genomes. Second, I have conducted deep evolutionary analyses to identify viral 

genes that have been repurposed for host function. And third, I have used cryogenic electron 

microscopy to determine the structure of viral defense systems in bacteria. 



1 

 

 

CHAPTER ONE 

INTRODUCTION 

Viruses are the most abundant biological entities on the planet1,2. As obligate intracellular 

genetic parasites, viruses require a host cell which they invade and reprogram to produce more 

virus. Viral progeny exit the reprogrammed host cell and remain biologically inert until they 

encounter a new host, at which point the viral replication cycle begins again. Since the discovery 

of the tobacco mosaic virus in 1898, viruses have been found to infect cells from every domain 

of life3–5. This selfish reproductive strategy is the most successful on the planet: there are an 

estimated 10 viral particles for every cell on earth1,2,6. Viruses influence life from the cellular 

level to the global level. An estimated 20-40% of the ocean’s biomass is lysed every day by viral 

infection, in a stunning illustration of how selfish genetic elements run riot in the world1.  

However, these estimates incompletely capture the extent to which selfish genetic 

elements dominate the biosphere. Some genetic parasites, viral-like elements called transposons 

and retrotransposons, embed in the host genome, replicate within the cell, and insert new copies 

directly into the genome of their original host. A surprising finding of the Human Genome 

Project was the discovery that approximately half of the human genome is made up of 

transposons, retrotransposons, and their decaying remnants7–9. Metagenomic studies indicate that 

selfish genetic elements may make up to 8% of bacterial genes10. In eukaryotes, selfish genetic 

elements can make up anywhere from 3% to 92% of the genome11,12. A survey of complete 

genomes and meta transcriptome data found that transposase genes are by far the most abundant 

genes in nature13. These proteins are used by selfish genetic elements to integrate selfish DNA 



2 

 

 

directly into the host genome13. Between extracellular viruses and intracellular genome parasites, 

selfish genetic elements exert tremendous pressure on their cellular hosts.  

Conserved Viral Defense Systems 

In response to the ubiquitous threat of selfish genetic elements, host cells across all 

domains of life have evolved diverse defense systems. Some defense systems are conserved from 

bacteria to eukaryotes and interfere with critical and universal steps in viral replication14–16. The 

cGAS/CD-NTase and STING pathways sense viral infection and synthesize cyclic di-nucleotide 

secondary messengers to activate an anti-viral response17–19. The viperin systems alter 

ribonucleotide pools in response to viral infection, terminating RNA synthesis20,21. Activation of 

gasdermins upon viral infection triggers assembly of membrane-puncturing pores leading to cell 

death before the replication cycle can complete22,23. Argonaute proteins use short nucleic-acid 

guides to target and cleave selfish RNA or DNA24,25. Toll/Interleukin-1 receptor (TIR) domains 

associated with pathogen-associated molecular pattern receptors deplete cellular NAD+ upon 

sensing a viral molecular pattern, depleting cellular energy26,27. Defense mechanisms conserved 

across the domains of life emphasize the ubiquity of viral parasitism.  They also underscore the 

truth in the saying coined by the inventor of the microscope, Antoni von Leeuwenhoek,  and 

famously paraphrased by Jacques Monod and François Jacob: “what is true for bacteria is also 

true for elephants.”28 

Unique Viral Defense Systems 

However, not everything that is true for bacteria is true for elephants. Some defense 

systems, such as the adaptive immune system of vertebrates, have no known homologues in 



3 

 

 

prokaryotes. Others, such as the CRISPR-Cas and restriction modification (RM) systems, have 

only been found in prokaryotes29,30. Studying these outliers have provided significant insights 

into the diversity of viral defense mechanisms and tools that have revolutionized medicine and 

molecular biology.  

 Early studies of bacterial immune systems revealed bacteria possess a mechanism to 

distinguish between self and non-self31,32. Now called restriction-modification (RM) systems, 

these two-component systems generally encode a methyl-transferase and a nuclease. Methylation 

of the genome creates a distinct signature of self. Invading nucleic acids from viruses are 

recognized and cleaved at specific target sites by nucleases, shutting down the viral infection33,34.  

In a clear example of ‘molecular arms race’35–38, viruses have evolved mechanisms to shut down 

these first-line of defense systems. For example, the “overcoming classical restriction” (Ocr) 

protein jams up restriction modification systems by mimicking viral DNA and blocking the 

active site of the restriction endonuclease.39 By blocking nuclease activity, viruses carrying Ocr 

circumvent this first-line defense system.  

CRISPR-Cas systems are prokaryote-specific immune systems that also distinguish 

between self and non-self. However, a key distinguishing feature of the CRISPR-Cas systems is 

that they are both adaptable and heritable. In CRISPR-Cas systems, clustered regularly 

interspaced short palindromic repeats (CRISPRs) catalogue foreign DNA to be read out by 

CRISPR-associated (Cas) proteins which target and degrade complementary foreign nucleic acid. 

40,41. Though structurally diverse, CRISPR-Cas systems generate adaptive immunity through 

three universally conserved steps: biogenesis, interference, and adaptation (reviewed in 42–45).  In 

CRISPRs, conserved direct repeat sequences flank unique virally-derived sequences called 



4 

 

 

spacers46–48. During biogenesis, Cas proteins process the transcribed CRISPR array into 

individual guide RNAs that contain a single unique spacer sequence49,50.  These guide RNAs are 

loaded into RNA-guided endonucleases that identify and cleave foreign nucleic acid during 

interference51,52 In the adaptation stage, Cas1 and Cas2 assemble into a heterohexameric 

complex that binds fragments of foreign nucleic acid and inserts them as new spacers through a 

transposition-like mechanism53.  

Abundance Leads to Co-option For Defense 

The Cas1 protein that mediates foreign DNA insertion derives from the transposase of an 

ancient selfish genetic element called a casposon51,54,55. Cas1 represents a striking example of 

how selfish genes may be repurposed for the host as ‘guns for hire’ 37,51,55. In CRISPR immunity, 

Cas1 performs a very similar function to its transposase ancestor, inserting short fragments of 

foreign DNA into the CRISPR by transposition53,56–59. However, Cas1 no longer services the 

replication of a selfish genetic element, but rather functions to protect the cellular host from 

selfish threats by providing the CRISPR interference machinery with updated guide RNAs.  

Intriguingly, adaptive immunity in eukaryotes also relies on a domesticated transposase. 

Vertebrates possess an adaptive immune system that is capable of generating molecular 

memories of pathogens60. However, most of vertebrate adaptive immunity is not heritable. 

Memories of infections are not transmitted from generation to generation, though they are 

preserved for months to years in the form of memory B cells. Rather, the V(D)J recombinase 

generates fragments of DNA that are ligated back together to create variable antigen recognition 

sites in antibodies in a mechanism that is quite similar to transposition54,61. Indeed, domestication 

of an ancient DNA transposon gave rise to this form of eukaryotic adaptive immunity. These two 



5 

 

 

striking examples of transposases domesticated for defense in prokaryotes and eukaryotes begs a 

deeper analysis of parasitic genes embedded within host genomes to identify parasites 

repurposed for host function.  

Domestication Beyond Defense 

In mammalian genomes, the most common class of selfish elements are retrotransposons, 

which generate new copies using a ‘copy-and-paste’ mechanism62–65. Retrotransposons are 

classified into two broad groups: long-terminal-repeat (LTR) retrotransposons and non-long-

terminal-repeat (non-LTR) retrotransposons, which include both autonomous LINE-1 

retrotransposons and non-autonomous Alu and SVA elements that rely on LINE-1 replication 

machinery. LTR retrotransposons (which include endogenous retroviruses, ERVs) encode 

structural group-specific antigen (gag) genes and enzymatic polymerase (pol) genes, flanked by 

LTRs. Their ‘exogenous’ retroviral counterparts also encode an envelope protein (env)66,67, which 

mediates membrane fusion needed for infection, and can subvert immune defenses via an 

immunosuppressive domain68.  

Multiple retroviral envelope genes have been domesticated for retroviral defense in 

mice69,70, humans71, sheep72, cats73,74, and chickens75. However, env genes have also been 

domesticated for non-defense function as a class of genes called syncitins. Syncitin proteins 

mediate membrane fusion to form multinucleated syncytial trophoblast cells in the placenta76,77. 

Syncytin gene domestication is a remarkable example of convergence, with at least seven 

independent events in different mammalian orders77,78, and in unusual lineages of lizard79 and 

fish80. These env domestications illustrate two key points. First, viral genes can be substrate for 



6 

 

 

novel host functions beyond their viral function. Second, the homologous viral domains may be 

repurposed for completely different functions.  

 Gag genes of LTR retrotransposons and retroviruses encode a polyprotein that includes 

capsid, matrix, and nucleocapsid domains that together package the viral genome during the 

virion assembly process65,81. Like env, gag has been domesticated both for retroviral restriction 

in mice (e.g., the Fv1 gene82–84), as well as for other critical host functions85–89. At least four 

domesticated capsid genes have been shown to assemble into capsid-like structures90–94, which 

perform essential functions in host reproduction87 and neuronal function90,95. One of these gag-

derived genes is vertebrate Arc (Activity-Regulated, Cytoskeletal-associated), which originated 

from an ancient LTR retrotransposon. ARC protein forms capsid-like structures and functions in 

the brain to regulate learning and memory as a signaling hub and messenger RNA shuttle in 

neurons90,95. An independent domestication event led to dArc1 in Drosophila species, which also 

forms capsid-like structures and packages mRNA for intercellular neuronal signaling92,96. 

Together with Cas1 and the V(D)J recombinase, Arc and dArc1 elegantly demonstrate how 

functional homologues from selfish genetic elements have been repurposed for similar host 

functions across massive evolutionary distances.   

Sequence and Structure 

Genetic conflict plays out in the ecosystem of genomes97. Two major technological 

advances have facilitated the study of these ecosystems by rendering previously invisible 

molecular processes visible. Genome sequences contain the set of instructions that build proteins, 

organelles, cells, and organisms. Fred Sanger changed the world when he developed his method 

for efficiently sequencing DNA98, because for the first time in human history, the genetic code 



7 

 

 

was readable. This has had profound implications for evolutionary biology, as it is now possible 

to reconstruct the evolutionary history of entire kingdoms of life from DNA sequence. It has also 

revolutionized our understanding of genetic conflict. The first full length genomes ever 

sequenced were the MS2 viral RNA genomes and the bacteriophage ØX174 genome98,99. The 

explosion of sequenced genomes has led to a corresponding explosion in the number of genetic 

parasites and defense systems against those parasites100. 

While genomic information provides the blueprint for a biological entity, structural 

techniques are critical for understanding the structure and function of the machines encoded by 

nucleic acid instructions. Here again, viruses have played a critical role in developing a new way 

of seeing. Because most viruses are not visible in the visible light spectrum, the development of 

new microscopy techniques was necessary to understand the physical basis for viral infections. 

Some of the first electron micrographs ever recorded were images of viral infections of 

bacteria101 and the first three-dimensional reconstruction from electron microscopy data was the 

reconstruction of a T4 bacteriophage tail102.  

Dissertation Overview  

This dissertation examines both sequence and structure to understand the evolutionary 

outcomes of genetic conflict. The first three chapters deal with the assembly and annotation of 

defense systems, genetic parasites, and domesticated genes in two complete, telomere-to-

telomere eukaryotic genomes. The fourth chapter begins to explore the functions beyond defense 

of domesticated genes in placental mammals. The final three chapters shift into the prokaryotic 

realm and use a combination of bioinformatics and cryogenic electron microscopy to study the 

ongoing molecular arms race in bacteria.  



8 

 

 

Chapter 2 presents telomere-to-telomere sequence of the genome of the algae SLA-04 

derived from long-read sequencing technology. Algae are a broad class of photosynthetic 

eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic 

diversity has been inferred from 18S rDNA sequencing since there are only a few complete 

genomes available in public databases. We used ultra-long-read Nanopore sequencing to 

determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, 

previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that 

grows to high cell density in a wide variety of environments – high and neutral pH, high and low 

alkalinity, and high and low salinity. SLA-04’s ability to grow in high pH and high alkalinity 

media without external CO2 supply is favorable for large-scale algal biomass production. 

Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences 

consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of 

Chlorella sorokiniana. We complement traditional genome annotation methods with high 

throughput structural predictions and demonstrate that this approach expands functional 

prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the 

genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for 

lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that 

may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust 

cultivation in mass production systems. Collectively, this work clarifies the phylogeny of 

Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can 

be used to improve annotation beyond sequence-based methods. 



9 

 

 

 Chapter 3 presents an analysis of the first human telomere-to-telomere genome in search 

of CRISPR-Cas adaptive immune systems. CRISPR-Cas systems are found in about 40% of 

bacterial and 85% of archaeal genomes, but not to date in eukaryotic genomes. Recently, a paper 

published in Communications Biology reported the identification of 12,572 putative CRISPRs in 

the human genome, which they call “hCRISPR”. We attempted to reproduce this analysis using 

the telomere-to-telomere human genome and show that repetitive elements identified as putative 

CRISPR loci contain neither the repeat-spacer-repeat architecture nor the cas genes characteristic 

of functional CRISPR systems.  

 Chapter 4 presents an in-depth evolutionary analysis of viral genes domesticated for host 

function in placental mammals. Using a complete human genome assembly and 25 additional 

vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of 

capsid genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our 

study expands on previous analyses to unearth several new insights about the evolutionary 

histories of these ancient genes. We find that at least five independent domestication events 

occurred from diverse Metaviridae, giving rise to three universally-retained single-copy genes 

evolving under purifying selection and two gene families unique to placental mammals with 

multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse 

amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its 

retention in several mammalian lineages, and differential utilization of an ancient programmed 

ribosomal frameshift in RTL3 between the domesticated capsid and protease domains. Our 

analyses also reveal that most members of the PNMA family in mammalian genomes encode a 

conserved putative amino-terminal RNA-binding domain both adjoining and independent from 



10 

 

 

domesticated capsid domains. Our analyses lead to a significant correction of previous 

annotations of the essential CCDC8 gene. We show that this putative RNA-binding domain is 

also present in several extant Metaviridae, revealing a novel protein domain configuration in 

retrotransposons. Collectively, our study reveals the divergent outcomes of multiple 

domestication events from diverse Metaviridae in the common ancestor of placental mammals.  

 Chapter 5 demonstrates that two PNMA family genes, PNMA1 and PNMA4, support 

reproductive capacity during aging. Analysis of donated human ovaries shows that expression of 

both genes declines normally with age, while several PNMA1 and PNMA4 variants identified in 

genome-wide association studies are causally associated with low testosterone, altered puberty 

onset, or obesity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from 

wild-type littermates, but by six months the mutant mice become prematurely subfertile, with 

precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they 

produce markedly fewer offspring than controls. These findings expand our understanding of 

factors that maintain human reproductive health and lend insight into the functional consequence 

of the domestication of retrotransposon-derived genes.  

Chapter 6, explains how viruses compete for limited cellular resources by delivering 

defense mechanisms that protect the host from competing genetic parasites1. PARIS is a defense 

system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 

35 kDa TOPRIM nuclease (AriB)2. However, the mechanism by which AriA and AriB function 

in phage defense is unknown. Here we show that AriA and AriB assemble into a 425 kDa 

supramolecular immune complex. We use cryo-EM to determine the structure of this complex 

which explains how six molecules of AriA assemble into a propeller-shaped scaffold that 



11 

 

 

coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the 

release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving 

host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that 

is not cleaved by PARIS, and thereby restores viral infection. Collectively, these data explain 

how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB 

toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes 

for the recognition of foreign proteins, rather than foreign nucleic acids3.   

Chapter 7 describes how bacteria and archaea acquire resistance to viruses and plasmids 

by integrating fragments of foreign DNA into the first repeat of a CRISPR array. The mechanism 

of site-specific integration in diverse CRISPR systems remains poorly understood. Here, we 

determine a 560 kDa integration complex structure that explains how Pseudomonas aeruginosa 

Cas (Cas1-2/3) and non-Cas proteins (integration host factor) fold 150 base-pairs of host DNA 

into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend 

traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR 

repeat in the Cas1 active site. Both Cas3s rotate 100-degrees to expose DNA binding sites on 

either side of the Cas2 homodimer, that each bind an inverted repeat motif in the leader. Leader 

sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5’-

GT. Collectively, this work reveals new DNA binding surfaces on Cas2 that are critical for DNA 

folding, and site-specific delivery of foreign DNA. 

In Chapter 8, I expand on the work of Chapter 7 by purifying the Cas1-2/3 fusion of 

Pseudomonas aeruginosa and determining multiple cryoEM structures at distinct stages of 

CRISPR adaptation. Collectively, these structures reveal a prominent channel on one face of the 



12 

 

 

integration complex that captures short fragments of foreign DNA. Foreign DNA binding 

triggers a series of conformational changes that reposition Cas3, exposing the Cas1 active sites 

and new DNA binding surfaces that are necessary for homing the DNA-bound integrase to the 

CRISPR locus. Upon docking to the CRISPR, the integrase catalyzes two sequential 

transesterification reactions that covalently link a discrete fragment of foreign dsDNA to the first 

repeat of the CRISPR locus. Taken together, these cryoEM structures clarify how the Cas1/2-3 

fusion proteins orchestrate foreign DNA capture, site specific delivery, and integration of new 

DNA into the bacterial genome. 

References 

1. Suttle, C. A. Viruses in the sea. Nature 437, 356–361 (2005). 

2. Hendrix, R. W., Smith, M. C. M., Burns, R. N., Ford, M. E. & Hatfull, G. F. Evolutionary 

relationships among diverse bacteriophages and prophages: All the world’s a phage. 

Proceedings of the National Academy of Sciences 96, 2192–2197 (1999). 

3. Torsvik, T. & Dundas, I. D. Bacteriophage of Halobacterium salinarium. Nature 248, 680–

681 (1974). 

4. d’Herelle, F. Sur un microbe invisible antagoniste des bacilles dysentétriques. Comptes 

rendus de l’Académie des sciences 165, 373–375 (1917). 

5. Beijerinck, M. W. On a Contagium vivum fluidum causing the Spotdisease of the Tobacco-

leaves. Koninklijke Nederlandse Akademie van Wetenschappen Proceedings Series B 

Physical Sciences 1, 170–176 (1898). 

6. Suttle, C. A. The significance of viruses to mortality in aquatic microbial communities. 

Microb Ecol 28, 237–243 (1994). 

7. Venter, J. C. et al. The Sequence of the Human Genome. Science 291, 1304–1351 (2001). 

8. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–

921 (2001). 

9. Li, W.-H., Gu, Z., Wang, H. & Nekrutenko, A. Evolutionary analyses of the human genome. 

Nature 409, 847–849 (2001). 



13 

 

 

10. Brazelton, W. J. & Baross, J. A. Abundant transposases encoded by the metagenome of a 

hydrothermal chimney biofilm. The ISME Journal 3, 1420–1424 (2009). 

11. Shao, C. et al. The enormous repetitive Antarctic krill genome reveals environmental 

adaptations and population insights. Cell 186, 1279-1294.e19 (2023). 

12. Kim, J. M., Vanguri, S., Boeke, J. D., Gabriel, A. & Voytas, D. F. Transposable elements 

and genome organization: a comprehensive survey of retrotransposons revealed by the 

complete Saccharomyces cerevisiae genome sequence. Genome Res 8, 464–478 (1998). 

13. Aziz, R. K., Breitbart, M. & Edwards, R. A. Transposases are the most abundant, most 

ubiquitous genes in nature. Nucleic Acids Research 38, 4207–4217 (2010). 

14. Culbertson, E. M. & Levin, T. C. Eukaryotic CD-NTase, STING, and viperin proteins 

evolved via domain shuffling, horizontal transfer, and ancient inheritance from prokaryotes. 

PLOS Biology 21, e3002436 (2023). 

15. Wein, T. & Sorek, R. Bacterial origins of human cell-autonomous innate immune 

mechanisms. Nat Rev Immunol 22, 629–638 (2022). 

16. Ledvina, H. E. & Whiteley, A. T. Conservation and similarity of bacterial and eukaryotic 

innate immunity. Nat Rev Microbiol 22, 420–434 (2024). 

17. Whiteley, A. T. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. 

Nature 567, 194–199 (2019). 

18. Wu, J. & Chen, Z. J. Innate Immune Sensing and Signaling of Cytosolic Nucleic Acids. 

Annual Review of Immunology 32, 461–488 (2014). 

19. Wu, J. et al. Cyclic GMP-AMP Is an Endogenous Second Messenger in Innate Immune 

Signaling by Cytosolic DNA. Science 339, 826–830 (2013). 

20. Bernheim, A. et al. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 

120–124 (2021). 

21. Rivera-Serrano, E. E. et al. Viperin Reveals Its True Function. Annual Review of Virology 7, 

421–446 (2020). 

22. Broz, P., Pelegrín, P. & Shao, F. The gasdermins, a protein family executing cell death and 

inflammation. Nat Rev Immunol 20, 143–157 (2020). 

23. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. 

Science 375, 221–225 (2022). 

24. Zaremba, M. et al. Short prokaryotic Argonautes provide defence against incoming mobile 

genetic elements through NAD+ depletion. Nat Microbiol 7, 1857–1869 (2022). 



14 

 

 

25. Anzelon, T. A. et al. Structural basis for piRNA targeting. Nature 597, 285–289 (2021). 

26. Ofir, G. et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. 

Nature 600, 116–120 (2021). 

27. Bayless, A. M. et al. Plant and prokaryotic TIR domains generate distinct cyclic ADPR 

NADase products. Science Advances 9, eade8487 (2023). 

28. Lane, N. The unseen world: reflections on Leeuwenhoek (1677) ‘Concerning little animals’. 

Philos Trans R Soc Lond B Biol Sci 370, 20140344 (2015). 

29. Koonin, E. V. Evolution of RNA- and DNA-guided antivirus defense systems in prokaryotes 

and eukaryotes: common ancestry vs convergence. Biol Direct 12, 5 (2017). 

30. Dimitriu, T., Szczelkun, M. D. & Westra, E. R. Evolutionary Ecology and Interplay of 

Prokaryotic Innate and Adaptive Immune Systems. Current Biology 30, R1189–R1202 

(2020). 

31. Arber, W., Hattman, S. & Dussoix, D. On the host-controlled modification of bacteriophage 

λ. Virology 21, 30–35 (1963). 

32. Arber, W. Host specificity of DNA produced by Escherichia coli. Journal of Molecular 

Biology 11, 247–256 (1965). 

33. Loenen, W. A. M., Dryden, D. T. F., Raleigh, E. A., Wilson, G. G. & Murray, N. E. 

Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids 

Research 42, 3–19 (2014). 

34. Bickle, T. A. & Krüger, D. H. Biology of DNA restriction. Microbiol Rev 57, 434–450 

(1993). 

35. Elde, N. C., Child, S. J., Geballe, A. P. & Malik, H. S. Protein kinase R reveals an 

evolutionary model for defeating viral mimicry. Nature 457, 485–489 (2009). 

36. Elde, N. C. & Malik, H. S. The evolutionary conundrum of pathogen mimicry. Nature 

Reviews Microbiology 7, 787–797 (2009). 

37. Koonin, E. V., Makarova, K. S., Wolf, Y. I. & Krupovic, M. Evolutionary entanglement of 

mobile genetic elements and host defence systems: guns for hire. Nature Reviews Genetics 

21, 119–131 (2020). 

38. Bernheim, A. & Sorek, R. The pan-immune system of bacteria: antiviral defence as a 

community resource. Nat Rev Microbiol 18, 113–119 (2020). 



15 

 

 

39. Krüger, D. H., Reuter, M., Hansen, S. & Schroeder, C. Influence of phage T3 and T7 gene 

functions on a type III (EcoP1) DNA restriction-modification system in vivo. Mol Gen 

Genet 185, 457–461 (1982). 

40. Lee, H. & Sashital, D. G. Creating memories: molecular mechanisms of CRISPR adaptation. 

Trends in Biochemical Sciences 47, 464–476 (2022). 

41. Koonin, E. V. & Makarova, K. S. Origins and evolution of CRISPR-Cas systems. Philos 

Trans R Soc Lond B Biol Sci 374, 20180087 (2019). 

42. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 

and derived variants. Nat Rev Microbiol 18, 67–83 (2020). 

43. Jackson, S. A. et al. CRISPR-Cas: Adapting to change. Science 356, eaal5056 (2017). 

44. Mohanraju, P. et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-

Cas systems. Science 353, aad5147 (2016). 

45. Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-Mediated Adaptive Immune Systems 

in Bacteria and Archaea. Annual Review of Biochemistry 82, 237–266 (2013). 

46. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short 

palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 

151, 2551–2561 (2005). 

47. Mojica, F. J. M., Díez-Villaseñor, C., Soria, E. & Juez, G. Biological significance of a family 

of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. 

Molecular Microbiology 36, 244–246 (2000). 

48. Jansen, Ruud., Embden, Jan. D. A. van, Gaastra, Wim. & Schouls, Leo. M. Identification of 

genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology 43, 

1565–1575 (2002). 

49. Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif 

sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 

155, 733–740 (2009). 

50. Brouns, S. J. J. et al. Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science 

321, 960–964 (2008). 

51. Krupovic, M., Makarova, K. S., Forterre, P., Prangishvili, D. & Koonin, E. V. Casposons: a 

new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic 

CRISPR-Cas immunity. BMC Biology 12, 36 (2014). 



16 

 

 

52. Haft, D. H., Selengut, J., Mongodin, E. F. & Nelson, K. E. A Guild of 45 CRISPR-

Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic 

Genomes. PLOS Computational Biology 1, e60 (2005). 

53. Nuñez, J. K., Lee, A. S. Y., Engelman, A. & Doudna, J. A. Integrase-mediated spacer 

acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015). 

54. Koonin, E. V. & Krupovic, M. Evolution of adaptive immunity from transposable elements 

combined with innate immune systems. Nat Rev Genet 16, 184–192 (2015). 

55. Krupovic, M., Béguin, P. & Koonin, E. V. Casposons: mobile genetic elements that gave rise 

to the CRISPR-Cas adaptation machinery. Current Opinion in Microbiology 38, 36–43 

(2017). 

56. Wright, A. V. et al. Structures of the CRISPR genome integration complex. Science 357, 

1113–1118 (2017). 

57. Santiago-Frangos, A., Buyukyoruk, M., Wiegand, T., Krishna, P. & Wiedenheft, B. 

Distribution and phasing of sequence motifs that facilitate CRISPR adaptation. Current 

Biology 31, 3515-3524.e6 (2021). 

58. Fagerlund, R. D. et al. Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR 

adaptation complex. Proceedings of the National Academy of Sciences 114, E5122–E5128 

(2017). 

59. Wiegand, T. et al. Reproducible Antigen Recognition by the Type I-F CRISPR-Cas System. 

The CRISPR Journal 3, 378–387 (2020). 

60. Flajnik, M. F. & Kasahara, M. Origin and evolution of the adaptive immune system: genetic 

events and selective pressures. Nat Rev Genet 11, 47–59 (2010). 

61. Liu, C., Zhang, Y., Liu, C. C. & Schatz, D. G. Structural insights into the evolution of the 

RAG recombinase. Nat Rev Immunol 22, 353–370 (2022). 

62. Krupovic, M. et al. Ortervirales: New Virus Order Unifying Five Families of Reverse-

Transcribing Viruses. Journal of Virology 92, e00515-18 (2018). 

63. Finnegan, D. J. Retrotransposons. Curr. Biol. 22, R432-437 (2012). 

64. Burns, K. H. & Boeke, J. D. Human Transposon Tectonics. Cell 149, 740–752 (2012). 

65. Dodonova, S. O., Prinz, S., Bilanchone, V., Sandmeyer, S. & Briggs, J. A. G. Structure of 

the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses. PNAS 116, 10048–

10057 (2019). 



17 

 

 

66. Doolittle, R. F., Johnson, M. S. & McClure, M. A. Origins and Evolutionary Relationships of 

Retroviruses. The Quarterly Review of Biology 64, 1–30 (1989). 

67. Hayward, A. Origin of the retroviruses: when, where, and how? Current Opinion in Virology 

25, 23–27 (2017). 

68. Ashkenazi, A., Faingold, O. & Shai, Y. HIV-1 fusion protein exerts complex 

immunosuppressive effects. Trends in Biochemical Sciences 38, 345–349 (2013). 

69. Ikeda, H., Laigret, F., Martin, M. A. & Repaske, R. Characterization of a molecularly cloned 

retroviral sequence associated with Fv-4 resistance. J Virol 55, 768–777 (1985). 

70. Taylor, G. M., Gao, Y. & Sanders, D. A. Fv-4: identification of the defect in Env and the 

mechanism of resistance to ecotropic murine leukemia virus. J Virol 75, 11244–11248 

(2001). 

71. Frank, J. A. et al. Evolution and antiviral activity of a human protein of retroviral origin. 

Science 378, 422–428 (2022). 

72. Varela, M., Spencer, T. E., Palmarini, M. & Arnaud, F. Friendly Viruses. Annals of the New 

York Academy of Sciences 1178, 157–172 (2009). 

73. Ito, J. et al. Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous 

Retroviruses. Journal of Virology 87, 12029–12040 (2013). 

74. Ito, J., Baba, T., Kawasaki, J. & Nishigaki, K. Ancestral Mutations Acquired in Refrex-1, a 

Restriction Factor against Feline Retroviruses, during its Cooption and Domestication. 

Journal of Virology 90, 1470–1485 (2016). 

75. Robinson, H. L., Astrin, S. M., Senior, A. M. & Salazar, F. H. Host Susceptibility to 

endogenous viruses: defective, glycoprotein-expressing proviruses interfere with infections. 

Journal of Virology 40, 745–751 (1981). 

76. Kim, F. J., Battini, J.-L., Manel, N. & Sitbon, M. Emergence of vertebrate retroviruses and 

envelope capture. Virology 318, 183–191 (2004). 

77. Dupressoir, A., Lavialle, C. & Heidmann, T. From ancestral infectious retroviruses to bona 

fide cellular genes: role of the captured syncytins in placentation. Placenta 33, 663–671 

(2012). 

78. Lavialle, C. et al. Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in 

placentation. Philosophical Transactions of the Royal Society B: Biological Sciences 368, 

20120507 (2013). 



18 

 

 

79. Cornelis, G. et al. An endogenous retroviral envelope syncytin and its cognate receptor 

identified in the viviparous placental Mabuya lizard. Proceedings of the National Academy 

of Sciences 114, E10991–E11000 (2017). 

80. Henzy, J. E., Gifford, R. J., Kenaley, C. P. & Johnson, W. E. An Intact Retroviral Gene 

Conserved in Spiny-Rayed Fishes for over 100 My. Molecular Biology and Evolution 34, 

634–639 (2017). 

81. Olson, E. D. & Musier-Forsyth, K. Retroviral Gag protein - RNA interactions: Implications 

for specific genomic RNA packaging and virion assembly. Semin Cell Dev Biol 86, 129–

139 (2019). 

82. Bénit, L. et al. Cloning of a new murine endogenous retrovirus, MuERV-L, with strong 

similarity to the human HERV-L element and with a gag coding sequence closely related to 

the Fv1 restriction gene. J Virol 71, 5652–5657 (1997). 

83. Yap, M. W., Colbeck, E., Ellis, S. A. & Stoye, J. P. Evolution of the Retroviral Restriction 

Gene Fv1: Inhibition of Non-MLV Retroviruses. PLOS Pathogens 10, e1003968 (2014). 

84. Young, G. R., Yap, M. W., Michaux, J. R., Steppan, S. J. & Stoye, J. P. Evolutionary journey 

of the retroviral restriction gene Fv1. PNAS 115, 10130–10135 (2018). 

85. Brandt, J., Veith, A.-M. & Volff, J.-N. A family of neofunctionalized Ty3/gypsy 

retrotransposon genes in mammalian genomes. Cytogenet. Genome Res. 110, 307–317 

(2005b). 

86. Campillos, M., Doerks, T., Shah, P. K. & Bork, P. Computational characterization of 

multiple Gag-like human proteins. Trends in Genetics 22, 585–589 (2006). 

87. Ono, R. et al. Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes 

early embryonic lethality. Nat Genet 38, 101–106 (2006). 

88. Sekita, Y. et al. Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal 

interface of mouse placenta. Nat Genet 40, 243–248 (2008). 

89. Kokošar, J. & Kordiš, D. Genesis and regulatory wiring of retroelement-derived 

domesticated genes: a phylogenomic perspective. Mol Biol Evol 30, 1015–1031 (2013). 

90. Pastuzyn, E. D. et al. The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag 

Protein that Mediates Intercellular RNA Transfer. Cell 172, 275-288.e18 (2018). 

91. Abed, M. et al. The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell 

lineage specification. PLOS ONE 14, e0214110 (2019). 

92. Erlendsson, S. et al. Structures of virus-like capsids formed by the Drosophila neuronal Arc 

proteins. Nat Neurosci 1–4 (2020) doi:10.1038/s41593-019-0569-y. 



19 

 

 

93. Segel, M. et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can 

be pseudotyped for mRNA delivery. Science 373, 882–889 (2021). 

94. Xu, J. et al. PNMA2 forms immunogenic non-enveloped virus-like capsids associated with 

paraneoplastic neurological syndrome. Cell 187, 831-845.e19 (2024). 

95. Nikolaienko, O., Patil, S., Eriksen, M. S. & Bramham, C. R. Arc protein: a flexible hub for 

synaptic plasticity and cognition. Seminars in Cell & Developmental Biology 77, 33–42 

(2018). 

96. Ashley, J. et al. Retrovirus-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic 

Boutons. Cell 172, 262-274.e11 (2018). 

97. Brookfield, J. F. Y. The ecology of the genome — mobile DNA elements and their hosts. 

Nat Rev Genet 6, 128–136 (2005). 

98. Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. 

Proc Natl Acad Sci U S A 74, 5463–5467 (1977). 

99. Fiers, W. et al. Complete nucleotide sequence of bacteriophage MS2 RNA: primary and 

secondary structure of the replicase gene. Nature 260, 500–507 (1976). 

100. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial 

pangenome. Science 359, eaar4120 (2018). 

101. Luria, S. E. & Anderson, T. F. The Identification and Characterization of Bacteriophages 

with the Electron Microscope. Proc Natl Acad Sci U S A 28, 127-130.1 (1942). 

102. De Rosier, D. J. & Klug, A. Reconstruction of Three Dimensional Structures from Electron 

Micrographs. Nature 217, 130–134 (1968). 

 

  



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CHAPTER TWO 

GENOME SEQUENCE, PHYLOGENETIC ANALYSIS, AND 

STRUCTURE BASED ANNOTATION REVEALS METABOLIC 

POTENTIAL OF CHLORELLA SP. SLA-04 

Contribution of Authors and Co-Authors 

Manuscript in Chapter 2 

Author: Calvin L.C. Goemann 

Contributions: Conceptualization, methodology, investigation & data collection, genomics & 

bioinformatics analysis, writing – original draft, writing – review & editing  

 

Co-Author: Royce Wilkinson 

Contributions: Conceptualization, methodology, investigation & data collection, writing – review 

& editing 

 

Co-Author: William Henriques  

Contributions: Genomics & bioinformatics analysis, writing – review & editing 

Co-Author: Huyen Bui 

Contributions: Genomics & bioinformatics analysis, writing – review & editing 

Co-Author: Hannah M. Goemann  

Contributions: Investigation & data collection, genomics & bioinformatics analysis, writing – 

review & editing 

 

Co-Author: Ross P. Carlson  

Contributions: Conceptualization, writing – review & editing 

Co-Author: Srihar Viamajala   

Contributions: Conceptualization, writing – review & editing 



21 

 

 

Co-Author: Robin Gerlach    

Contributions: Conceptualization, writing – review & editing 

Co-Author: Blake Wiedenheft  

Contributions: Conceptualization, methodology, writing – original draft, writing – review & 

editing 

  



22 

 

 

Manuscript Information 

Calvin L. C. Goemann, Royce Wilkinson, William Henriques, Huyen Bui, Hannah M. Goemann, 

Ross P. Carlson, Sridhar Viamajala, Robin Gerlach, Blake Wiedenheft 

 

Algal Research 

Status of Manuscript:  

☐ Prepared for submission to a peer-reviewed journal 

☐ Officially submitted to a peer-reviewed journal 

☐ Accepted by a peer-reviewed journal 

☑ Published in a peer-reviewed journal 

 

Elsevier  

Volume 69 

January 2023  

 

Submitted: 17 February 2022 

Published online: 13 December 2022 

https://doi.org/10.1016/j.algal.2022.102943 

  

https://doi.org/10.1016/j.algal.2022.102943


23 

 

 

Abstract 

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and 

physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA 

sequencing since there are only a few complete genomes available in public databases. Here we 

use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete 

genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-

04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of 

environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04’s 

ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for 

large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and 

conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage 

from other strains of Chlorella sorokiniana. We complement traditional genome annotation 

methods with high throughput structural predictions and demonstrate that this approach expands 

functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies 

the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for 

lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that 

may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust 

cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella 

sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to 

improve annotation beyond sequence-based methods. 

  



24 

 

 

Introduction 

Algae are diverse photosynthetic eukaryotes that assimilate six times more nitrogen than 

terrestrial plants, perform 45% of global oxygenic photosynthesis, and 50% of total CO2 fixation1–

4. The phylogenetic and physiological diversity of algae is mirrored by the diversity of aquatic 

ecosystems where algae are found (e.g., temperatures 0-56oC, pH 0-11.5, and salt concentrations 

0.02-3M)5,6. However, it is currently unclear how selective pressures from disparate aquatic 

environments shape algal genotypes and corresponding phenotypes.  

Genomic analysis of algae across different environments is essential to understand how 

algae survive and adapt to changing conditions. As of September 2022, there are 170 green algae 

genomes in the National Center for Biotechnology Information (NCBI) database. Most of these 

genomes are highly fragmented and only 16 contain defined chromosome sequences. This resource 

limitation restricts efforts to confidently identify genotypes that drive functional diversity. 

Understanding the phylogenetic and functional diversity of algae will benefit from a broader 

collection of complete algal genomes that can be used for comparative analyses.  

To date, most algal genomes have been sequenced using short-read sequencing 

platforms7,8. While these platforms are accurate, PCR bias and short read lengths frequently result