A cohesin-RAD21 interactome.

The cohesin complex holds the sister chromatids together from S-phase until the metaphase-to-anaphase transition, and ensures both their proper cohesion and timely separation. In addition to its canonical function in chromosomal segregation, cohesin has been suggested by several lines of investigation in recent years to play additional roles in apoptosis, DNA-damage response, transcriptional regulation and haematopoiesis. To better understand the basis of the disparate cellular functions of cohesin in these various processes, we have characterized a comprehensive protein interactome of cohesin-RAD21 by using three independent approaches: Y2H (yeast two-hybrid) screening, immunoprecipitation-coupled-MS of cytoplasmic and nuclear extracts from MOLT-4 T-lymphocytes in the presence and absence of etoposide-induced apoptosis, and affinity pull-down assays of chromatographically purified nuclear extracts from pro-apoptotic MOLT-4 cells. Our analyses revealed 112 novel protein interactors of cohesin-RAD21 that function in different cellular processes, including mitosis, regulation of apoptosis, chromosome dynamics, replication, transcription regulation, RNA processing, DNA-damage response, protein modification and degradation, and cytoskeleton and cell motility. Identification of cohesin interactors provides a framework for explaining the various non-canonical functions of the cohesin complex.

Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species.

The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega<1), approximately 3.7% of ompC codon sites in E. aerogenes (omega=21.52) are under the influence of positive Darwinian selection (i.e. omega>1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.

Sensitivity of RECQL4-deficient fibroblasts from Rothmund-Thomson syndrome patients to genotoxic agents.

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund-Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage.

Adaptive evolution after duplication of penaeidin antimicrobial peptides.

Penaeidin antimicrobial peptides in penaeid shrimps are an important component of their innate immune system that provides immunity against infection caused by several gram-positive bacteria and filamentous fungal species. Despite the knowledge on the identification and characterization of these peptides in penaeid shrimps, little is known about the evolutionary pattern of these peptides and the underlying genetic mechanisms that maintain high sequence diversities in the penaeidin gene family. Based on the phylogenetic analyses and maximum likelihood-based codon substitution analyses, here we present the convincing evidence that multiple copies of penaeidins have evolved by gene duplication, and positive Darwinian selection (adaptive evolution) is the likely cause of accelerated rate of amino acid substitutions among these duplicated genes. While the average ratio of non-synonymous to synonymous substitutions (omega) for the entire coding region of both active domains is 0.9805, few codon sites showed significantly higher omega (3.73). The likelihood ratio tests that compare models incorporating positive selection (omega>1) at certain codon sites with models not incorporating positive selection (omega<1), failed to reject (p=0) the evidence of positive Darwinian selection. The rapid adaptive evolution of this gene family might be directed by the pathogens and the faster rate of amino acid substitutions in the N-terminal proline-rich and C-terminal cysteine-rich domains could be due to their direct involvement in the protection against pathogens. When the host expose to different habitats/environment an accelerated rate of amino acid substitutions in both the active domains may also be expected.

Pattern of nucleotide substitution and divergence of prophenoloxidase in decapods.

Despite the unprecedented development in identification and characterization of prophenoloxidase (proPO) in commercially important decapods, little is known about the evolutionary relationship, rate of amino acid replacement and differential selection pressures operating on proPO of different species of decapods. Here we report the evolutionary relationship among these nine decapod species based on proPO gene and types of selective pressures operating on proPO codon sites. Our analyses revealed that all the nine decapod species shared a common ancestor. The mean percentage sequence divergence at proPO gene was 34.4+/-0.6%. Pairwise estimates of nonsynonymous to synonymous ratio (omega) for Homarus americanus-H. gammarus is greater than one, therefore indicating adaptive evolution (functional diversification) of proPO in these two species. In contrast, strong purifying selection (omega<1) was observed in all other species pairs. However, phylogenetically closely related decapods revealed relatively higher omega value (omega=0.15+/-0.3) than the distantly related species pairs (omega=0.0075+/-0.005). These discrepancies could be due to higher fixation probability of beneficial mutation in closely related species. Maximum likelihood-based codon substitution analyses revealed a strong purifying selection operating on most of the codon sites, therefore suggesting proPO is functionally constrained (purifying selection). Codon substitution analyses have also revealed the evidence of strong purifying selection in haemocyanin subunits of decapods.

Positive Darwinian selection on crustacean hyperglycemic hormone (CHH) of the green shore crab, Carcinus maenas.

The tissue-specific expression and differential function of the crustacean hyperglycemic hormone (CHH) in Carcinus maenas indicate an interesting evolutionary history. Previous studies have shown that CHH from the sinus gland X-organ (XO-type) has hyperglycemic activity, whereas the CHH from the pericardial organ (PO-type) neither shows hyperglycemic activity nor it inhibits Y-organ ecdysteroid synthesis. Here we examined the types of selective pressures operating on the variants of CHH in Carcinus maenas. Maximum likelihood-based codon substitution analyses revealed that the variants of this neuropeptide in C. maenas have been subjected to positive Darwinian selection indicating adaptive evolution and functional divergence among the CHH variants leading to two unique groups (PO and XO-type). Although the average ratio of nonsynonymous to synonymous substitution (omega) for the entire coding region is 0.5096, few codon sites showed significantly higher omega (10.95). Comparison of models that incorporate positive selection (omega > 1) with models not incorporating positive selection (omega <1) at certain codon sites failed to reject (p=0) evidence of positive Darwinian selection.

The occurrence of Vibrio species in tropical shrimp culture environments; implications for food safety.

The occurrence of various Vibrio species in water, sediment and shrimp samples from multiple shrimp farm environments from the east and west coast of India was studied. The relative abundance was higher in west coast farms (ca. 10(4) cfu/ml water) when compared to the east coast (ca. 10(2) cfu/ml water). Vibrio alginolyticus (3-19%), V. parahaemolyticus (2-13%), V. harveyi (1-7%) and V. vulnificus (1-4%) were the predominant Vibrio species identified by standard biochemical testing. In some cases, V. cholerae could be found, but all isolates were negative for the cholera toxin (ctx) gene that is associated with choleragenic strains. The biochemical identification of V. parahaemolyticus, the other human pathogen among the species mentioned above, was confirmed by PCR targeting the toxR gene and a 387 bp chromosomal locus specific for this species. Furthermore, the presence of the virulence-associated tdh (thermostable direct haemolysin) and trh (TDH-related haemolysin) genes in the V. parahaemolyticus isolates was also detected by PCR. Only 2 out of 47 isolates were tdh positive and one contained the trh gene. However, since V. cholerae, V. parahaemolyticus and V. vulnificus species are recognized as a major cause of seafood-borne illness, it is important to pay attention to post-harvest handling and adequate cooking.

Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses in hatchery-reared Penaeus monodon postlarvae.

The prevalence of hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in samples of Penaeus monodon postlarvae (PL10 to PL20, 10 to 20 d old postlarvae) in India was studied by PCR. Samples collected from different hatcheries, and also samples submitted by farmers from different coastal states, were analyzed. HPV was detected in 34%) of the hatchery samples and 31% of the samples submitted by farmers, using a primer set designed for detection of HPV from P. monodon in Thailand. However, none of these samples were positive using primers designed for detection of HPV from P. chinensis in Korea. This indicated that HPV from India was more closely related to HPV from P. monodon in Thailand. MBV was detected in 64% of the samples submitted by the farmers and 71% of the hatchery samples. A total of 84 % of the samples submitted by farmers, and 91% of the hatchery samples, were found positive for WSSV. Prevalence of concurrent infections by HPV, MBV and WSSV was 27% in hatchery samples and 29%, in samples submitted by farmers. Only 8% of the hatchery samples and 16% of the samples submitted by farmers were negative for all 3 viruses. This is the first report on the prevalence of HPV in P. monodon postlarvae from India.

Multiple viral infection in Penaeus monodon shrimp postlarvae in an Indian hatchery.

Moribund Penaeus monodon postlarvae (PL8-PL10) in a hatchery in India were found to be simultaneously infected by 3 different viruses. They were highly infected with monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) by histology and with white spot syndrome virus (WSSV) by non-nested polymerase chain reaction (PCR). Apparently healthy postlarvae tested from the same hatchery were positive for MBV and WSSV by nested PCR only. Tissue sections of such postlarvae did not show any histopathological changes. The simultaneous occurrence of these 3 viruses in hatchery-reared postlarval P. monodon is being reported for the first time.