Evidence Of Homologous Recombination Research Articles

Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.

CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.

Real-world (RW) use of niraparib (Nir) monotherapy (mono) as first-line (1L) switch maintenance (1LSM) therapy following chemotherapy (CT) plus bevacizumab (bev) induction treatment in patients (pts) with epithelial ovarian cancer (EOC) in the SW1TCH study.

536 Background: In the RW CHAR1ZMA study, 414 pts with EOC treated with 1L platinum-based CT (pbCT) and receiving 1L maintenance (1LM) nir mono had a median time to next treatment (TTNT) from end of 1L, a proxy for RW progression-free survival, of 13.3 mo (95% CI, 12.0–15.8 mo).1 Limited data exist on RW outcomes in pts with EOC who receive nir mono as 1LSM after 1L pbCT + bev treatment. The SW1TCH study describes nir mono use and outcomes as 1LSM therapy after 1L pbCT + bev treatment in a US RW setting. Methods: Using the nationwide Flatiron Health electronic health record–derived deidentified database, this retrospective observational study included pts ≥18 y of age at initial EOC diagnosis who received 1L pbCT + bev treatment followed by nir 1LM mono, initiated between Jan 1, 2017, and Sep 2, 2022. The index date was the initiation of nir 1LM. Pts were followed until the earliest of date of death, last activity, or end of study (Nov 30, 2022). Time to treatment discontinuation (TTD) and TTNT were estimated with Kaplan-Meier methods. Results: Of 93 pts selected, median age at index was 67 y (IQR, 60–72 y). Most pts had an Eastern Cooperative Oncology Group performance score of 0–1 (75.3%) and stage III/IV disease at diagnosis (89.2%). Overall, 86.0% of pts had BRCA wild-type EOC. 19.4% and 22.6% of pts had evidence of homologous recombination (HR)-deficient and HR-proficient EOC, respectively, whereas HR deficiency status was unknown for 58.1%. Median duration of 1L therapy was 4.8 mo (IQR, 3.6–6.0 mo), and median time from end of 1L pbCT to start of 1LM was 1.2 mo (IQR, 0.8–1.8 mo). Reasons for nir discontinuation are shown in the Table. Median TTD from index was 9.3 mo (95% CI, 6.0–11.3 mo). Median TTNT from end of 1L was 12.9 mo (95% CI, 11.5–19.0 mo). Conclusions: Observed results are consistent with CHAR1ZMA,1 suggesting the RW benefit of nir mono in 1LM regardless of bev use with1L pbCT. This RW study demonstrates that nir mono as 1LSM therapy is a viable treatment option for pts with advanced EOC.

Identification of Garlic virus A infecting Allium sativum L. through next generation sequencing technology

ABSTRACT Garlic virus A (GarV-A) severely damages and reduces the yield of economically important garlic crop worldwide. Here, we present the first comprehensive view of GarV-A infecting Yamuna Safed-3 (G282) of garlic (Allium sativumL.) variety from India at the complete genome sequence level. RNA-seq was performed on an Illumina HiSeq 2000 platform to obtain the full length GarV-A genome and analysed by different software and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the presence of the GarV-A. The virus complete genome consists of 8,793 nucleotides (nt), excluding the poly (A) tail at the 3ʹ terminus, with 5ʹ and 3ʹ untranslated regions (UTRs) of 193 and 123 nt, respectively. Genome analysis of GarV-A Indian isolate shared higher nucleic acid and amino acid sequence identity to Chinese isolates than to Australian and Spanish isolates. Further RDP analysis showed that the Indian GarV-A (MT731489) isolate was found to be recombinant with most part of the RNA segment is derived from China GarV-E isolate (MN059326) as the minor parent and the Russia ShV-X (JX310755) isolate as the major parent. More research is needed to determine the outspread of this particular recombinant virus. This is the first report of the complete genome sequence and evidence of homologous recombination in of GarV-A from India.

Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5′ C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5′ C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

Evidence of homologous recombination as a driver of diversity in Brachyspira pilosicoli.

The enteric, pathogenic spirochaete Brachyspira pilosicoli colonizes and infects a variety of birds and mammals, including humans. However, there is a paucity of genomic data available for this organism. This study introduces 12 newly sequenced draft genome assemblies, boosting the cohort of examined isolates by fourfold and cataloguing the intraspecific genomic diversity of the organism more comprehensively. We used several in silico techniques to define a core genome of 1751 genes and qualitatively and quantitatively examined the intraspecific species boundary using phylogenetic analysis and average nucleotide identity, before contextualizing this diversity against other members of the genus Brachyspira . Our study revealed that an additional isolate that was unable to be species typed against any other Brachyspira lacked putative virulence factors present in all other isolates. Finally, we quantified that homologous recombination has as great an effect on the evolution of the core genome of the B. pilosicoli as random mutation (r/m=1.02). Comparative genomics has informed Brachyspira diversity, population structure, host specificity and virulence. The data presented here can be used to contribute to developing advanced screening methods, diagnostic assays and prophylactic vaccines against this zoonotic pathogen.

Genetic characterization and recombination analysis of atypical porcine pestivirus

Atypical porcine pestivirus (APPV) is recognised as the etiology of congenital tremor (CT) Type A-II and poses a challenge to pig production. Here, we described a CT case in piglets caused by APPV infection in central China in 2017. Interestingly, different from a previous report, more CT litters were observed in the second and third parity sows compared to the first and fourth parity. Evolutionary analysis and recombination evaluation were conducted for the isolate and 61 APPV genomes were available in GenBank. Phylogenetic analysis revealed a high level of genetic variation of APPV and the coexistence of three clades (Clades I–III) in China. The isolate was clustered into Clade I, which seemed to be prevalent worldwide and displayed higher genetic variability (Subgroups 1–4) compared with Clade II and Clade III, both of which were only reported in China. Notably, three putative recombinants were identified and characterized in APPV. The recombination events occurred in inter-clades (Clade II and III) or intra-clades (Clade I). To the best of our knowledge, this study presents the first evidence of homologous recombination within Pestivirus K. These results provide new clinical presentations of APPV infection and may be helpful in better understanding the large amount of genetic variations in this genus.

Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.

Homologous recombination (HR) is considered a major driving force of evolution because it generates and expands genetic diversity. Evidence of HR between coinfecting herpesvirus DNA genomes can be found frequently both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when coinfecting viral genomes interact. To study the spatiotemporal requirements for intergenomic recombination, we developed an assay with dual-color FISH that enables detection of HR between different pairs of coinfecting HSV-1 genomes. Our results revealed that HR increases intermingling of RCs derived from different genomes. Furthermore, inhibition of RC movement reduces the rate of HR events among coinfecting viruses. Finally, we observed correlation between nuclear size and the number of RCs per nucleus. Our findings suggest that both viral replication and recombination are subject to nuclear spatial constraints. Other DNA viruses and cellular DNA are likely to encounter similar restrictions.-Tomer, E., Cohen, E. M., Drayman, N., Afriat, A., Weitzman, M. D., Zaritsky, A., Kobiler, O. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.

Whole-Genome Sequencing of Human Clinical Klebsiella pneumoniae Isolates Reveals Misidentification and Misunderstandings of Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae.

Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. K.variicola and K.quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K.pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K.pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K. variicola and K.quasipneumoniae. Whole-genome sequencing of 95 additional non-ESBL-producing K.pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K.pneumoniae, suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K.variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella. IMPORTANCEKlebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K.variicola and K.quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K.variicola and K. quasipneumoniae isolates are often misidentified as K.pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K.pneumoniae. The presence of KPC in K.variicola and K.quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K.variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Abstract 2476: Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models

Abstract Rucaparib (CO-338) is an oral small molecule inhibitor of the poly(ADP-ribose) polymerase (PARP) enzymes PARP-1, PARP-2 and PARP-3 that is being developed for cancer patients with evidence of homologous recombination (HR) deficiency, including BRCA1 and BRCA2 mutations. PARP inhibitors have demonstrated clinical activity in BRCA1, BRCA2, and ATM mutant prostate cancer patients (Mateo et al. N Engl J Med. 2015;373:1697-708); however, limited preclinical validation of PARP inhibitors in prostate cancer has been reported to date, since no BRCA mutant nor HR deficient prostate cell lines have been identified. In this study, we modelled the functional inactivation of DNA repair genes using siRNA and CRISPR/Cas9, and examined the impact on PARP inhibition, in the DU145 and PC-3 prostate cancer cell lines. These cell lines were chosen based on transfection efficiency, lack of obvious genetic defects in DNA repair pathways, and baseline insensitivity to rucaparib (IC50 > 10 µM). A panel of 26 DNA repair genes were individually knocked down by siRNA, and changes to rucaparib potency were examined in a 7-day cell viability assay. The knockdown of BRCA1 or BRCA2 increased rucaparib cytotoxicity in both prostate cell lines. For example, the IC50 of rucaparib in DU145 and PC-3 cells with reduced BRCA2 levels was 275 and 297 nM, representing a 36- and 37-fold increase in rucaparib sensitivity, respectively. In addition to BRCA1 and BRCA2, the knockdown of several other genes including BARD1, CDK12, FANCA, PALB2, RAD51, RAD51C, RAD51D, and RAD54L increased rucaparib efficacy ≥ 2-fold in at least 1 cell line examined. To further investigate HR deficiency in prostate cancer, BRCA2 was knocked out in the human 22Rv1 prostate cancer cell line using CRISPR/Cas9. Clone 1-50 displayed biallelic BRCA2 inactivation due to the introduction of a deleterious premature stop codon, and immunoblot analysis verified lack of the full-length BRCA2 protein. The IC50 of rucaparib in the parental and clone 1-50 cells was 9,504 and 287 nM, respectively, demonstrating a 33-fold increase in rucaparib cytotoxicity in this BRCA2 deficient model. Androgen receptor (AR) signaling and proliferation were also examined in clone 1-50. In contrast to published reports (Schiewer et al. Cancer Discov. 2012;2:1134-49), no impact of PARP inhibition on dihydrotestosterone (DHT) induced gene expression was observed in the parental 22Rv1 and BRCA2 deficient clone 1-50 cell lines. In vivo model development and efficacy studies with the 22Rv1 and clone 1-50 cell lines are ongoing. Taken together, these findings support the hypothesis that deficiencies in BRCA1 and BRCA2, as well as additional HR genes, may sensitize prostate cancer cells to rucaparib. A phase 2 study investigating rucaparib in patients with metastatic castration-resistant prostate cancer and HR gene deficiency (TRITON2) is ongoing (NCT02952534). Citation Format: Minh Nguyen, Andrew D. Simmons, Thomas C. Harding. Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2017-2476

Evidence of recombination in Tula virus strains from Serbia

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

A Model for the Effect of Homologous Recombination on Microbial Diversification

The effect of homologous recombination (HR) on the evolution of microbial genomes remains contentious as competing hypotheses seek to explain the evolutionary dynamics of microbial species. Evidence for HR between microbial genomes is widespread, and this process has been proposed to act as a cohesive force that can constrain the diversification of microbial lineages. We seek to characterize the evolutionary dynamics of sympatric populations to explore the impact of HR on microbial speciation. We describe a simple equation for quantifying the cohesive effect of HR on microbial populations as a function of their nucleotide divergence, μ/ρ = πg10 − 20 πg. The model was verified using a forward-time microbial population simulator that can explore the evolutionary dynamics of sympatric populations in nonoverlapping niche space. The model was also evaluated using multilocus sequence data from a range of microbial species, providing criteria for dividing them into either cohesively recombining or clonally diverging lineages. We conclude that models of microbial diversification that appear contradictory can be explained in a unified manner as the natural and predictable consequence of variation in a small number of population parameters.

Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91-100% and 70-98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.

SNP and haplotype identification of the wheat monomeric α-amylase inhibitor genes

Seventy-three gene sequences encoding monomeric alpha-amylase inhibitors were characterized from cultivated wheat "Chinese Spring", group 6 nullisomic-tetrasomic lines of "Chinese Spring" and diploid putative progenitors of common wheat. The monomeric alpha-amylase inhibitors from the different sources shared very high homology (99.54%). The different alpha-amylase inhibitors, which were determined by the 24 single nucleotide polymorphisms (SNPs) of their gene sequences, were investigated. A total of 15 haplotypes were defined by sequence alignment, among which 9 haplotypes were found with only one single sequence sample. Haplotype H02 was found to be the main haplotype occurring in 83 WMAI sequence samples, followed by haplotype H11. The median-joining network for the 15 haplotypes of monomeric alpha-amylase inhibitor gene sequences from hexaploid wheats was star like, and at least two subclusters emerged. Furthermore evidence of homologous recombination was found between the haplotypes. The relationship between nucleotide substitutions and the amino acid changes in WMAI of hexaploid wheats was summarized. It was clear that only five polymorphic sites in the nucleotide sequence of WMAI resulted in amino acid variations, and that should be the reason for different structure and function of inhibitors. However, little evidence could be found that there were WMAI genes in the A genome of hexaploid wheat, whereas it could conclude from our results that the A genome diploid wheat had WMAI genes. The overall information on the monomeric alpha-amylase inhibitors from wheat and Aegilops strongly support the view that these inhibitors have evolved from a common ancestral gene through duplication and mutation.

Homologous recombination and the pattern of nucleotide substitution in Ehrlichia ruminantium

Patterns of nucleotide substitution at orthologous loci were examined between three genomes of Ehrlichia ruminantium, the causative agent of heartwater disease of ruminants. The most recent common ancestor of two genomes (Erwe and Erwo) belonging to the Welgevonden strain was estimated to have occurred 26,500–57,000 years ago, while the most recent common ancestor of these two genomes and the Erga genome (Gardel strain) was estimated to have occurred 2.1–4.7 million years ago. The search for genes showing extremely high values of the number of synonymous substitutions per site was used to identify genes involved in past homologous recombination. The most striking case involved the map1 gene, encoding major antigenic protein-1; evidence for homologous recombination is consistent with previous phylogenetic analysis of map1 alleles. At this and certain other loci, homologous recombination may have contributed to the evolution of host–pathogen interactions. In addition, comparison of the patterns of synonymous and nonsynonymous substitution provided evidence for positive selection favoring a high level of amino acid change between the Welgevonden and Gardel strains at a locus of unknown function (designated Erum4340 in the Erwo genome).

The human Y chromosome’s azoospermia factor b (AZFb) region: sequence, structure, and deletion analysis in infertile men

Microdeletions of the Y chromosome long arm are the most common mutations in infertile males, where they involve one or more “azoospermia factors” (AZFa, b, and c). Understanding of the...

The quality of merC, a module of the mer mosaic.

We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.

Complete nucleotide sequence of the antithrombin gene: Evidence for homologous recombination causing thrombophilia

Antithrombin is the principle regulator of thrombin and other blood coagulation proteinases. It is a member of the serpin family of proteinase inhibitors. The genomic sequence of the antithrombin locus has been completed, revealing a gene spanning 13,477 base pairs from the transcription start site to the poly(A) addition signal. Nine complete and one partial Alu repeat elements were identified within the introns of the gene, with all but one orientated in the reverse direction. Inherited deficiency of antithrombin is associated with a venous thrombotic tendency. Restriction fragment mapping of the antithrombin genes in an individual with type I antithrombin deficiency identified an intragenic deletion in one allele. Localization of the deletion breakpoints involved restriction analysis and direct sequencing of amplified DNA spanning the deletion site. The deletion removed 2761 base pairs, affecting exon 5 and flanking introns, with the deletion ends contained within the left components of two Alu elements. It is likely, therefore, that the deletion arose by homologous recombination between the two Alu elements.

Evidence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA

Closely linked genes for 18S and 5S rRNAs have been located on four different cloned SalI restriction fragments of wheat (Triticum aestivum L.) mitochondrial DNA. Restriction analysis has revealed that in each of the cloned fragments, the 18S and 5S rRNA genes are contained within the same basic structural unit, R, which is at least 4 kbp long. This unit is flanked by sequences designated u (0.8 kbp), v (13.7 kbp), w (0.7 kbp), and y (1.4 kbp), in the orientations v-R-w, v-R-y, u-R-w, and u-R-y in the four different SalI fragments. We conclude that 18S + 5S rRNA genes are located at several distinct sites in the wheat mitochondrial genome, and suggest that reciprocal intra- and/or intermolecular recombination between such repeated sequences could promote extensive genomic rearrangement and thus contribute to the physical heterogeneity that is a hallmark of most plant mitochondrial DNAs.

Hybrid gamma 2b-gamma 2a genes expressed in myeloma variants: evidence for homologous recombination.

The expressed immunoglobulin heavy chain genes of five gamma 2b-gamma 2a hybrid chain-producing variants of the mouse myeloma MPC-11 (gamma 2b, kappa) have been characterized by genomic Southern blot analysis. Results show that a hybrid gamma 2b-gamma 2a gene was formed in each variant by recombination between the expressed gamma 2b gene of MPC-11 and a gamma 2a gene. The recombination sites are within regions of marked homology between gamma 2b and gamma 2a genes: at least three and probably four variants show gamma 2b-gamma 2a recombination within the heavy chain constant region 2 (CH2) domain, while the fifth has its recombination site between the penultimate nucleotide of CH1 and the eighth nucleotide of the hinge. An unexpected finding is that the hybrid heavy chain-producing variants fall into two subgroups based on their use of different gamma 2a gene forms in hybrid gene formation. This result leads to the speculation that either a tandem gamma 2a gene duplication was present in MPC-11 prior to variant generation or mitotic recombination between chromosomes occurred in the generation of one variant subgroup. The similarity of hybrid gene formation in MPC-11 variants to that apparently responsible for concerted evolution within multigene families and hybrid protein expression in various individuals is noted, and the possible relationship between hybrid gene formation and the heavy chain class switch is discussed.