Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin - An immunohistochemical survey of 476 primary and metastatic carcinomas

CDX2 is a recently cloned homeobox gene that encodes an intestine-specific transcription factor, expressed in the nuclei of epithelial cells throughout the intestine, from duodenum to rectum. While expression of CDX2 protein in primary and metastatic colorectal carcinomas has been previously documented, neither the sensitivity nor the specificity of CDX2 expression, as determined by immunohistochemistry, for colorectal adenocarcinoma has been determined. We performed an immunohistochemical survey of 476 tumors with a monoclonal antibody, CDX2-88, including 89 tumors from the colon and duodenum and 95 tumors from other gastrointestinal sites, including the esophagus, stomach, pancreatobiliary system, gastrointestinal carcinoids, and liver. CDX2 was expressed uniformly (that is, in 76-100% of tumor cells) in all but one of the evaluated colorectal and duodenal tumors. High-level expression of CDX2 was also found, however, in mucinous ovarian carcinomas and adenocarcinomas primary to the urinary bladder of which 64% and 100% were positive, respectively. Gastric, gastroesophageal, and pancreatic adenocarcinomas and cholangiocarcinomas all showed similar, heterogeneous patterns of CDX2 expression. Most tumors in each group showed CDX2 expression by a minority of cells, whereas a substantial minority of cases in each group was completely negative and a smaller minority was uniformly positive. Gastrointestinal carcinoids gave similarly varied results, but the majority (58%) was negative. Hepatocellular carcinomas showed no expression of CDX2. Only very rare examples of carcinomas of the genitourinary and gynecologic tracts, breast, lung, and head and neck showed significant levels of CDX2 expression. In this study of primary and metastatic epithelial tumors, uniform CDX2 expression is demonstrated to be an exquisitely sensitive and highly, but incompletely, specific marker of intestinal adenocarcinomas. Compared with villin, a previously described marker of GI adenocarcinomas, CDX2 demonstrated superior sensitivity and comparable specificity. CDX2 expression can be seen, however, in selected non-GI adenocarcinomas such as mucinous ovarian carcinomas and adenocarcinomas of the urinary bladder.

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q(2). Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q(6), the coq10 mutant has near normal amounts of Q(6) in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the aromatic-rich protein family Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q(6)/mol of protein. We propose that Coq10p is a Q(6)-binding protein and that in the coq10 mutant Q(6) it is not able to act as an electron carrier, possibly because of improper localization.

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31 kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation. (C) 2004 Elsevier B.V. All rights reserved.

Sensitive Affimer and Antibody Based Impedimetric Label-Free Assays for C-Reactive Protein

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. Levels greater than 3 mu g/mL serum have hitherto been considered to indicate pathology, but there is increasing interest in assessments between 0.1 and 10 mu g/mL, which have been found to correlate with severity of risk for cardiovascular disease. We report herein the generation of both antibody and Affimer based impedance immunoassays for CRP that are substantially more sensitive than clinically utilized immunonephelometry and immunoturbidity assessments. Significant in this study is not only the use of a constrained peptide to detect a clinically important target but also that derived electrochemical impedance assays can be highly sensitive even with probes whose relatively weak (mu M) affinities are not amenable to target detection by surface plasmon resonance (SPR). Key to this finding is acknowledging that receptive surfaces of comparatively low initial steric bulk and charge transfer resistance are especially primed to be highly responsive to target binding in electroanalytical assays of this type.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.