Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

扩增共有序列遗传标记(ACGM)及其在植物中的应用价值

扩增共有序列遗传标记(Amplified consensus genetic markers, ACGM)是建立在亲缘关系较近的物种间同源基因中编码序列(外显子)存在高度保守性, 而非编码区(内含子)存在潜在多态性基础之上的一种基于PCR的新型DNA分子标记技术。随着比较基因组学和生物信息学的迅猛发展, ACGM技术已成为物种间同源基因比较、系统发生关系分析和感兴趣基因定位的有效工具。文章就ACGM技术尤其是它在芸薹属和禾本科物种中的应用研究做详细介绍。同时, 对ACGM技术可能的应用前景进行了展望。     

Refining the genetic portrait of Portuguese Roma through X-chromosomal markers

Due to differences in transmission between X-chromosomal and autosomal DNA, the comparison of data derived from both markers allows deeper insight into the forces that shape the patterns of genetic diversity in populations. In this study, we applied this comparative approach to a sample of Portuguese Roma (Gypsies) by analyzing 43 X-chromosomal markers and 53 autosomal markers. Portuguese individuals of non-Gypsy ancestry were also studied. Compared with the host population, reduced levels of diversity on the X chromosome and autosomes were detected in Gypsies; this result was in line with known patterns of genetic diversity typical of Roma groups. As a consequence of the complex demographic past of the Roma, during which admixture and genetic drift played major roles, the amount of linkage disequilibrium (LD) on the X chromosome in Gypsies was considerably higher than that observed in non-Gypsies. When the pattern of differentiation on the X chromosome was compared with that of autosomes, there was evidence for asymmetries in female and male effective population sizes during the admixture between Roma and non-Roma. This result supplements previous data provided by mtDNA and the Y chromosome, underlining the importance of using combined information from the X chromosome and autosomes to dissect patterns of genetic diversity. Following the out-of-India dispersion, the Roma acquired a complex genetic pattern that was influenced by drift and introgression with surrounding populations, with important contributions from both males and females. We provide evidence that a sex-biased admixture with Europeans is probably associated with the founding of the Portuguese Gypsies.     

Evaluating the genetic structure of wild and commercial red cusk-eel (Genypterus chilensis) populations through the development of novel microsatellite markers from a reference transcriptome

Molecular Biology Reports - The red cusk-eel (Genypterus chilensis) is a native Chilean species with a high-value market, with the potential to diversify Chilean aquaculture. The objective of this...     

Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome

Background

Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.

Methodology/Principal Findings

Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.

Conclusions/Significance

These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.     

Improved breast cancer prognosis through the combination of clinical and genetic markers

MOTIVATION: Accurate prognosis of breast cancer can spare a significant number of breast cancer patients from receiving unnecessary adjuvant systemic treatment and its related expensive medical costs. Recent studies have demonstrated the potential value of gene expression signatures in assessing the risk of post-surgical disease recurrence. However, these studies all attempt to develop genetic marker-based prognostic systems to replace the existing clinical criteria, while ignoring the rich information contained in established clinical markers. Given the complexity of breast cancer prognosis, a more practical strategy would be to utilize both clinical and genetic marker information that may be complementary. METHODS: A computational study is performed on publicly available microarray data, which has spawned a 70-gene prognostic signature. The recently proposed I-RELIEF algorithm is used to identify a hybrid signature through the combination of both genetic and clinical markers. A rigorous experimental protocol is used to estimate the prognostic performance of the hybrid signature and other prognostic approaches. Survival data analyses is performed to compare different prognostic approaches. RESULTS: The hybrid signature performs significantly better than other methods, including the 70-gene signature, clinical makers alone and the St. Gallen consensus criterion. At the 90% sensitivity level, the hybrid signature achieves 67% specificity, as compared to 47% for the 70-gene signature and 48% for the clinical makers. The odds ratio of the hybrid signature for developing distant metastases within five years between the patients with a good prognosis signature and the patients with a bad prognosis is 21.0 (95% CI:6.5-68.3), far higher than either genetic or clinical markers alone. AVAILABILITY: The breast cancer dataset is available at www.nature.com and Matlab codes are available upon request.     

Investigations on the variability of four genetic serum protein markers in Poland.

198 unrelated male and female Poles from Ostrów Wielkopolski (Central Poland) and 228 unrelated male and female Kashubes from Ko?cierzyna (Northern Poland) have been typed for four polymorphic serum protein systems: HP, TF, GC, and PI. Phenotype and allele frequencies of all these four polymorphic systems are quite different between Poles and Kashubes. Comparisons with some other Central and East European population samples (Slovaks, Hungarians, Matyos, Gypsies) revealed a considerable genetic heterogeneity among them. Genetic distance analysis showed that Hungarians and Matyos as well as Poles and Slovaks are found in two subclusters, which are linked up to one cluster. Gypsies and especially Kashubes exhibit a distinct position from this cluster. This genetic distance pattern can be explained satisfactorily considering the ethnohistory of the population groups under study.     

Assignment of non-informative turkey genetic markers through comparative approaches

Molecular markers such as microsatellites, provide genetic signposts for navigating genomes. In general, genetic markers that are monomorphic or non-informative in mapping populations typically remain unmapped and as such are less likely to be included in future studies. The use of hybrid cell panels and in silico mapping via whole genome sequences allow for positional mapping of non-segregating markers. This study utilizes the INRA ChickRH6 whole-genome radiation hybrid panel and chicken whole-genome shotgun sequence to map microsatellite markers from the turkey (Meleagris gallopavo). Thirty-three of the 41 markers typed on the RH panel had significant linkage to at least one other marker and 83 of 100 sequences returned significant BLAST similarities. Positioning of these markers provides additional sequence tagged sites in the turkey genome and increases the potential use of these markers for future genetic studies.     

Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.     

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.     

Evaluation of the genetic diversity of microsatellite markers among four strains of Oreochromis niloticus

Different strains of Nile tilapia can be found worldwide. To successfully use them in breeding programs, they must be genetically characterized. In this study, four strains of Nile tilapia – UFLA, GIFT, Chitralada and Red‐Stirling – were genetically characterized using 10 noncoding microsatellite loci and two microsatellites located in the promoter and first intron of the growth hormone gene (GH). The two microsatellites in the GH gene were identified at positions ?693 to ?679 in the promoter [motif (ATTCT)₈] and in intron 1 at positions +140 to +168 [motif (CTGT)₇]. Genetic diversity was measured as mean numbers of alleles and expected heterozygosity, which were 4 and 0.60 (GIFT), 3.5 and 0.71 (UFLA), 4.5 and 0.57 (Chitralada) and 2.5 and 0.42 (Red‐Stirling) respectively. Genetic differentiation was estimated both separately and in combination for noncoding and GH microsatellites markers using Jost's D_EST index. The UFLA and GIFT strains were the least genetically divergent (D_EST = 0.10), and Chitralada and Red‐Stirling were the most (D_EST = 0.58). The UFLA strain was genetically characterized for the first time and, because of its unique origin and genetic distinctness, may prove to be an important resource for genetic improvement of Nile tilapia. This study shows that polymorphisms found in coding gene regions might be useful for assessing genetic differentiation among strains.