Expression Of Transcript Variants Research Articles

Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse

In the male germ line, sequence-specific methylation patterns are initially acquired prenatally in diploid gonocytes and are further consolidated after birth during spermatogenesis. It is still unclear how DNA methyltransferases are involved in establishing and/or maintaining these patterns in germ cells, or how their activity is regulated. We compared the temporal expression patterns of the postulated de novo DNA methyltransferases DNMT3a and DNMT3b in murine male germ cells. Mitotic, meiotic and post-meiotic male germ cells were isolated, and expression of various transcript variants and isoforms of Dnmt3a and Dnmt3b was examined using Quantitative RT-PCR and Western blotting. We found that proliferating and differentiating male germ cells were marked by distinctive expression profiles. Dnmt3a2 and Dnmt3b transcripts were at their highest levels in type A spermatogonia, decreased dramatically in type B spermatogonia and preleptotene spermatocytes and rose again in leptotene/zygotene spermatocytes, while Dnmt3a expression was mostly constant, except in type B spermatogonia where it increased. In all cases, expression declined as pachynema progressed. At the protein level, DNMT3a was the predominant isoform in type B spermatogonia, while DNMT3a2, DNMT3b2, and DNMT3b3 were expressed throughout most of spermatogenesis, except in pachytene spermatocytes. We also detected DNMT3a2 and DNMT3b2 in round spermatids. Taken together, these data highlight the tightly regulated expression of these genes during spermatogenesis and provide evidence that DNMTs may be contributing differentially to the establishment and/or maintenance of methylation patterns in male germ cells.

β‐Catenin splice variants and downstream targets as markers for neoplastic progression of esophageal cancer

This study characterizes the frequency of exon 3 CTNNB1 mutations and compares the expression of CTNNB1 transcript variants and downstream targets MYC and WAF1 (p21) across the neoplastic progression of esophageal squamous cell carcinomas (ESCCs). Mutational analysis was performed on 56 tumors and corresponding germline DNA, using primers to exon 3 of CTNNB1 and SSCP DNA sequencing gels. Quantitative Real Time RT-PCR was performed on 45 foci representing the histological spectrum from normal to invasive cancer, using specific primer sets for alternative splice variants that differ by the presence (16A) or absence (16B) of a 159-bp noncoding segment of exon 16 of CTNNB1, in conjunction with downstream targets MYC and WAF1. Two unique mutations were identified, S37F in the SxxxS repeat region, and a germline polymorphism, T59A. Thus, mutation of CTNNB1 exon 3 is a rare event in this population. RT-PCR analysis successfully confirmed the presence of both beta-catenin splice variants in histologically normal and preneoplastic squamous epithelium, and invasive tumors of the esophagus, and identified a significant reduction in the 16A/16B ratio (P = 0.014) and an accompanying significant increase in the MYC/WAF1 expression ratio (P = 0.001) with progression from normal mucosa to dysplasia. This represents the first identification of two CTNNB1 transcripts in histologically "normal" esophageal squamous cells, squamous dysplasia, and invasive ESCC. These results show an increase in the minor mRNA (16B) isoform and changes in the expression of downstream markers consistent with increased transcription during the histological progression from normal to squamous dysplasia.

Elevated expression of survivin-splice variants predicts a poor outcome for soft-tissue sarcomas patients

The aim of this study was to investigate the level and the prognostic value of the expression of different survivin transcript variants--survivin, survivin-DeltaEx3 and survivin-2B--in tumours of 76 soft tissue sarcoma (STS) patients. The expression of survivin transcript variants in STS tissue samples and in 12 nonmalignant control tissues was analysed by quantitative RT-PCRs. Expression levels of all survivin transcript variants were strongly elevated in STS compared to normal tissues. A positive correlation between expression of splice variants and tumour stage was found (P=0.02; chi2 test). The multivariate Cox's proportional hazards regression model revealed a 7.3-fold increased risk of tumour-related death for patients with survivin-DeltaEx3 overexpressing tumours (P=0.007). The effect of surivivin (wildtype variant) and survivin-2B was less pronounced but still significant (2.2- and 1.9-fold, resp., P<0.05 each). Our results show for the first time that mRNA expression of survivin-variants is significantly correlated to a poor prognosis for STS patients, and we suggest expression of survivin splice variants together with tumour stage as independent predictor of survival.

Oocyte-specific gene signaling and its regulation of mammalian reproductive potential

Oocyte-specific genes play important roles in regulating ovarian development, principally through the proper and timely progression of oogenesis and folliculogenesis. Development of transgenic mouse models has been critical in revealing how oocyte-specific transcripts influence oocyte development and growth, integrity of the oocyte-granulosa cell complex, oocyte maturation, fertilization, and early embryonic development. Oocyte-derived genes that mediate recombination of homologous chromosomes and DNA mismatch repair include Spo11, Atm, Dmc1, Msh5, Mlh1, and Msh4. Transcripts such as Dazla and Fig-alpha regulate initial proliferation of the primordial germ cell and follicle. Transition from the primordial to primary follicle relies on the expression of growth factors bFGF, Gdf9 and Bmp15, as well as on the expression of various transcripts that mediate oocyte-granulosa cell interactions. Oocyte growth is predominantly under exogenous control, however resumption of meiotic progression is dictated by genes that influence proper chromatin and spindle regulation, such as Cdk, Histone H1oo, Fmn-2, Mad2, and Bub3. Maintenance of meiotic metaphase II arrest prior to fertilization is mediated primarily by c-mos, and successful fertilization requires the expression of zona pellucida glycoproteins (Zp1, Zp2, and Zp3) and Cd9. Following fertilization, maternal-effect and maternally expressed 'imprinting' genes are necessary for the completion of meiosis and for patterning early embryonic development. Recent utilization of suppressive subtractive hybridization (SSH), PCR amplification, and cDNA microarray analysis techniques alongside established transgenesis models are expanding the classification of novel oocyte-specific genes required for reproductive fitness in various species, including human.

Gene expression profiling of cultured human islet preparations.

The expression of functional and regulatory genes by islet cells is a key determinant for the success of islet transplantation. The aim of this study is twofold: first, to characterize the cluster of genes expressed in human islet isolations; and second, to validate the capability of gene array technology to assess with accuracy the expression of various transcripts. RNA from isolated islet preparations obtained from three independent donors was converted to cDNA and then transcribed to cRNA. Individual cRNA preparations were then hybridized to U133A microarrays carrying approximately 23,000 genes, and analyzed using GeneSpring (SiliconGenetics, Redwood City, CA) software. Real-time reverse transcription-polymerase chain reaction was performed to validate results obtained by microarray analysis. Microarray analysis identified the expression of about 7,000 genes transcribed in cultured human islet preparations. Enzymes represented the most abundant class of genes identified, followed by nuclear binding proteins, signal transduction molecules, transport proteins, and growth factor receptors and their ligands. Real-time polymerase chain reaction confirmed the identification of various islet-specific genes detected by microarray analysis, but also showed that such genes as pancreatic duodenal homeobox 1 protein and glucagon-like peptide 1 receptor, which were not detected by gene array, can be readily identified and quantified. In addition, gene array produced a suboptimal quantification of genes expressed in large amounts by islet cells. Indeed, the abundance of mRNA for insulin when compared with the level of somatostatin mRNA was not as different as one would have predicated based on the classic knowledge of islet physiology. Gene array analysis appears to be a valuable tool to obtain preliminary information of genes expressed by a given tissue. The expression levels of transcripts expressed in very low or very high quantities need to be confirmed by an independent technique.

Characterization of human mesothelin transcripts in ovarian and pancreatic cancer.

BackgroundMesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors.MethodsHuman genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts.ResultsIn silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA.ConclusionMesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin.

Respiratory pathway enzymes are differentially altered in flood tolerant and sensitive rice types during O 2 deprivation stress and post-stress recovery phase

O 2 deprivation stress leads to pronounced alterations in respiratory metabolism. The transcript level and specific activity of enzymes involved in glycolysis and ethanolic fermentation have previously been shown to be up-regulated in response to this stress. In this study, we compared transcript and protein levels of pyruvate decarboxylase (PDC) and transcript levels of alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI), phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), enolase (ENO) and pyruvate kinase (PK) in flood-sensitive IR54 and flood-tolerant FR13A rice types. The samples were obtained during the stress period as well as in the post-stress recovery phase. The level of constitutive expression of various transcripts was comparable in IR54 and FR13A rice types. The levels of PDC2 subunit appeared higher in shoots of FR13A relative to IR54 during stress and recovery. The Western profile of PDC showed an additional band in FR13A that was not apparent in IR54. During the recovery phase, IR54 shoots maintained higher levels of gpi, pfk, tpi, gapdh, eno, pk and pdc1 gene transcripts as compared with FR13A. On the other hand, adh1 gene transcript levels were relatively higher in FR13A rice at the 6 h stress and 3 h recovery stages. It thus appears that both transcriptional and post-transcriptional controls play a part in the response of contrasting rice types to O 2 deprivation stress.

The Roles of Individual Growth Factors on Differentiation

Embryonic stem (ES) cell research has a long way to go if it will someday provide us with replacement organs or tissues. One problem is not knowing what growth factors influence ES cells. Another dilemma is figuring out how to effect the differentiation of ES cells into desired differentiated cell lineages. Schuldiner et al . studied aggregates of ES cells called embryoid bodies and the effects of several growth factors on their differentiation. By analyzing the expression of various transcripts known to be markers for specific cell differentiation, the authors were able to categorize the effects of growth factor treatment into three groups: differentiation into mesodermal-like cells was induced by activin A or transforming growth factor-β; formation of cells with both mesodermal and ectodermal characteristics was induced by retinoic acid (RA), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or bone morphogenetic protein-4 (BMP-4); and formation of cells with characteristics of mesoderm, ectoderm, and endoderm was caused by nerve growth factor (NGF) and hepatocyte growth factor (HGF). However, the application of single growth factors to ES cells did not cause full differentiation into one specific cell type. Thus, although this work represents a first attempt at determining the effects of growth factors on pluripotent cells, other variables essential for proper differentiation, including the timing and impinging of multiple growth factors onto ES cells, will have to be accounted for in future studies. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D.A., and Benvenisty, N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc . Natl . Acad . Sci . U . S . A . 97 : 11307-11312. [Abstract] [Full Text]

The Roles of Individual Growth Factors on Differentiation

Embryonic stem (ES) cell research has a long way to go if it will someday provide us with replacement organs or tissues. One problem is not knowing what growth factors influence ES cells. Another dilemma is figuring out how to effect the differentiation of ES cells into desired differentiated cell lineages. Schuldiner et al. studied aggregates of ES cells called embryoid bodies and the effects of several growth factors on their differentiation. By analyzing the expression of various transcripts known to be markers for specific cell differentiation, the authors were able to categorize the effects of growth factor treatment into three groups: differentiation into mesodermal-like cells was induced by activin A or transforming growth factor-β; formation of cells with both mesodermal and ectodermal characteristics was induced by retinoic acid (RA), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or bone morphogenetic protein-4 (BMP-4); and formation of cells with characteristics of mesoderm, ectoderm, and endoderm was caused by nerve growth factor (NGF) and hepatocyte growth factor (HGF). However, the application of single growth factors to ES cells did not cause full differentiation into one specific cell type. Thus, although this work represents a first attempt at determining the effects of growth factors on pluripotent cells, other variables essential for proper differentiation, including the timing and impinging of multiple growth factors onto ES cells, will have to be accounted for in future studies.Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D.A., and Benvenisty, N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 97: 11307-11312.[Abstract][Full Text]