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1. Genome Size Changes by Duplication, Divergence, and Insertion in Caenorhabditis Worms

   https://doi.org/10.1093/molbev/msad039  

   Adams, Paula E; Eggers, Victoria K; Millwood, Joshua D; Sutton, John M; Pienaar, Jason; Fierst, Janna L (March 2023, Molecular Biology and Evolution)

   Arkhipova, Irina (Ed.)

   Abstract Genome size has been measurable since the 1940s but we still do not understand genome size variation. Caenorhabditis nematodes show strong conservation of chromosome number but vary in genome size between closely related species. Androdioecy, where populations are composed of males and self-fertile hermaphrodites, evolved from outcrossing, female-male dioecy, three times in this group. In Caenorhabditis, androdioecious genomes are 10–30% smaller than dioecious species, but in the nematode Pristionchus, androdioecy evolved six times and does not correlate with genome size. Previous hypotheses include genome size evolution through: 1) Deletions and “genome shrinkage” in androdioecious species; 2) Transposable element (TE) expansion and DNA loss through large deletions (the “accordion model”); and 3) Differing TE dynamics in androdioecious and dioecious species. We analyzed nematode genomes and found no evidence for these hypotheses. Instead, nematode genome sizes had strong phylogenetic inertia with increases in a few dioecious species, contradicting the “genome shrinkage” hypothesis. TEs did not explain genome size variation with the exception of the DNA transposon Mutator which was twice as abundant in dioecious genomes. Across short and long evolutionary distances Caenorhabditis genomes evolved through small structural mutations including gene-associated duplications and insertions. Seventy-one protein families had significant, parallel decreases across androdioecious Caenorhabditis including genes involved in the sensory system, regulatory proteins and membrane-associated immune responses. Our results suggest that within a dynamic landscape of frequent small rearrangements in Caenorhabditis, reproductive mode mediates genome evolution by altering the precise fates of individual genes, proteins, and the phenotypes they underlie. 

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2. Dual-targeting by CRISPR/Cas9 leads to efficient point-mutagenesis but only rare targeted deletions in the rice genome

   Pathak, Bhuvan; Zhao, Shan; Manoharan, Muthusamy; Srivastava, Vibha (March 2019, 3 biotech)

   The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion–deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended. 

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3. A Pilot Study of Blood-Based Methylation Markers Associated With Pancreatic Cancer

   https://doi.org/10.3389/fgene.2022.849839  

   Jansen, Rick J.; Orr, Megan; Bamlet, William R.; Petersen, Gloria M. (March 2022, Frontiers in Genetics)

   Over the past several decades in the United States, incidence of pancreatic cancer (PCa) has increased, with the 5-year survival rate remaining extremely low at 10.8%. Typically, PCa is diagnosed at an advanced stage, with the consequence that there is more tumor heterogeneity and increased probability that more cells are resistant to treatments. Risk factors for PCa can serve as a way to select a high-risk population and develop biomarkers to improve early detection and treatment. We focus on blood-based methylation as an approach to identify a marker set that can be obtained in a minimally invasive way (through peripheral blood) and could be applied to a high-risk subpopulation [those with recent onset type 2 diabetes (DM)]. Blood samples were collected from 30 patients, 15 had been diagnosed with PCa and 15 had been diagnosed with recent onset DM. HumanMethylationEPIC Beadchip (Illumina, CA, United States) was used to quantify methylation of approximately 850,000 methylation sites across the genome and to analyze methylation markers associated with PCa or DM or both. Exploratory analysis conducted to propose importance of top CpG (5′—C—phosphate—G—3′) methylation site associated genes and visualized using boxplots. A methylation-based age predictor was also investigated for ability to distinguish disease groups from controls. No methylation markers were observed to be significantly associated with PCa or new onset diabetes compared with control the respective control groups. In our exploratory analysis, one methylation marker, CpG04969764, found in the Laminin Subunit Alpha 5 ( LAMA5 ) gene region was observed in both PCa and DM Top 100 methylation marker sets. Modification of LAMA5 methylation or LAMA5 gene function may be a way to distinguish those recent DM cases with and without PCa, however, additional studies with larger sample sizes and different study types (e.g., cohort) will be needed to test this hypothesis. 

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4. Local Genomic Instability of the SpTransformer Gene Family in the Purple Sea Urchin Inferred from BAC Insert Deletions

   https://doi.org/10.3390/genes15020222  

   Barela_Hudgell, Megan A; Momtaz, Farhana; Jafri, Abiha; Alekseyev, Max A; Smith, L Courtney (February 2024, Genes)

   The SpTransformer (SpTrf) gene family in the purple sea urchin, Strongylocentrotus purpuratus, encodes immune response proteins. The genes are clustered, surrounded by short tandem repeats, and some are present in genomic segmental duplications. The genes share regions of sequence and include repeats in the coding exon. This complex structure is consistent with putative local genomic instability. Instability of the SpTrf gene cluster was tested by 10 days of growth of Escherichia coli harboring bacterial artificial chromosome (BAC) clones of sea urchin genomic DNA with inserts containing SpTrf genes. After the growth period, the BAC DNA inserts were analyzed for size and SpTrf gene content. Clones with multiple SpTrf genes showed a variety of deletions, including loss of one, most, or all genes from the cluster. Alternatively, a BAC insert with a single SpTrf gene was stable. BAC insert instability is consistent with variations in the gene family composition among sea urchins, the types of SpTrf genes in the family, and a reduction in the gene copy number in single coelomocytes. Based on the sequence variability among SpTrf genes within and among sea urchins, local genomic instability of the family may be important for driving sequence diversity in this gene family that would be of benefit to sea urchins in their arms race with marine microbes. 

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5. Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators

   https://doi.org/10.1371/journal.pgen.1011306  

   Gifford, Isaac; Suárez, Gabriel A; Barrick, Jeffrey E (September 2024, PLOS Genetics)

   Didelot, Xavier (Ed.)

   Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-freeAcinetobacter baylyistrain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulatorsrnd(ribonuclease D),csrA(RNA-binding carbon storage regulator), andhfq(RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restoredcsrAfunction. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments. 

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