Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.     

Identification of key genes involved in root development of tomato using expressed sequence tag analysis

Root system of plants are actually fascinating structures, not only critical for plant development, but also important for storage and conduction. Due to its agronomic importance, identification of genes involved in root development has been a subject of intense study. Tomato is the one of the most consumed vegetables in the world. Tomato has been used as model system for dicot plants because of its small genome, well-established transformation techniques and well-constructed physical map. The present study is targeted to identify of root specific genes expressed temporally and also gene(s) involved in lateral root and profuse root development. A total of 890 ESTs were identified from five EST libraries constructed using SSH approach which included temporal gene regulation (early and late) and genes involved in morphogenetic traits (lateral and profuse rooting). One hundred sixty-one unique ESTs identified from various libraries were categorized based on their putative functions and deposited in NCBI-dbEST database. In addition, 36 ESTs were selected for validation of their expression by RT-PCR. The present findings will help in shedding light to the unexplored developmental process of root growth in tomato and plant in general.     

Identification and validation of genes involved in gastric tumorigenesis

Background  

Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

Identification of genes involved in terbinafine resistance in Aspergillus nidulans

AIMS: To determine the pattern and the genetic basis of resistance to terbinafine, a drug extensively used for the treatment of fungal infections in humans. METHODS AND RESULTS: Four resistant mutants from Aspergillus nidulans isolated after irradiation with ultraviolet light were crossed with the master strain F (MSF). Genetic analysis revealed that a single gene, located on chromosome IV, is responsible for resistance to terbinafine and that the alleles responsible for this resistance in these mutants are of a codominant or dominant nature at high terbinafine concentrations. Furthermore, the interaction of this mutation with another one identified on chromosome II causes the double mutant to be highly resistant. CONCLUSIONS: Periodic surveillance of antimycotic susceptibility would be an important measure in detecting the emergence and spread of resistance. Mutation in a single gene could be responsible for resistance to terbinafine and a genic interaction may be responsible for a higher level of antimycotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of the mechanisms that lead to changes in the sensitivity of a fungus to a given antifungal agent is important both in order to define strategies for the use of such agent and to guide the development of new antifungal agents.     

Large-scale screen for genes involved in gonad development

The vertebrate gonad develops from the intermediate mesoderm as an initially bipotential organ anlage, the genital ridge. In mammals, Sry acts as a genetic switch towards testis development. Sox9 has been shown to act downstream of Sry in testis development, while Dax1 appears to counteract Sry. Few more genes have been implicated in early gonad development. However, the genetic networks controlling early differentiation events in testis and ovary are still far from being understood. In order to provide a broader basis for the molecular analysis of gonad development, high-throughput gene expression analysis was utilized to identify genes specifically expressed in the gonad. In total, among 138 genes isolated which showed tissue specific expression in the embryo, 79 were detected in the developing gonad or sex ducts. Twenty-seven have not been functionally described before, while 40 represent known genes and 12 are putative mouse orthologues. Forty-five of the latter two groups (86%) have not been described previously in the fetal gonad. In addition, 21 of the gonad specific genes showed sex-dimorphic expression suggesting a role in sex determination and/or gonad differentiation. Eighteen of the latter (86%) have not been described previously in the fetal gonad. In total we provide new data on 72 genes which may play a role in gonad or sex duct development and/or sex determination. Thus we have generated a large gene resource for the investigation of these processes, and demonstrate the suitability of high-throughput gene expression screening for the genetic analysis of organogenesis.     

Identification of Bradyrhizobium nod genes involved in host-specific nodulation.

Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes. Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species. lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product. A mutation in one of the loci was found to result in an alteration of the host range of B. japonicum. This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.     

Identification of genes involved in alcohol consumption and cigarettes smoking

We compared the results of quantitative linkage analysis using single-nucleotide polymorphisms and microsatellite markers and introduced a new screening test for multivariate quantitative linkage analysis using the Collaborative Study on the Genetics of Alcoholism data. We analyzed 115 extended non-Hispanic White families and tested for linkage using two phenotypes: the maximum number of drinks in a 24-hour period and the number of packs smoked per day for one year. Our results showed that the linkage signal increased using single-nucleotide polymorphisms compared with microsatellite markers and that the screening test gave similar results to that of the bivariate analysis, suggesting its potential use in reducing overall analysis time.     

Identification of Brucella spp. genes involved in intracellular trafficking

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago–lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn 5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis . We also examined the intracellular fate of three virB mutants ( virB2 , virB4 and virB9 ) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago–lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61β protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.     

Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes.