Qiang Fu

Mouse phosphoglycerate mutase M and B isozymes: cDNA cloning, enzyme activity assay and mapping.

Two mouse cDNAs encoding the non-muscle-specific or brain isoform (type B, Pgam1) and the muscle-specific isoform (type M, Pgam2) of phosphoglycerate mutase (PGAM) were isolated and characterized. Pgam1 contains a 765 bp open reading frame (ORF) coding for a 254-residue protein while Pgam2 contains a 762 bp ORF coding for a 253-residue protein. The deduced proteins of mouse Pgam1 and Pgam2 are highly similar to those of human and rat (> or = 93% similarity). Northern blot analysis showed that the expression patterns of Pgam1 and Pgam2 were distinct. Pgam1 was expressed as a 2.1-kb transcript highly in brain and kidney and moderately in liver, thyroid, stomach and heart, whereas Pgam2 was expressed as a 1.0-kb transcript highly in muscle, testis and moderately in heart and lung, but was not detectable in the other six tissues examined. Transfecting the cDNA fragments containing the entire ORFs of these two cDNAs into COS7 cells for transient expression, respectively, the enzyme activities of mouse Pgam1 and Pgam2 were detected to be 2.2-2.5 times of those of COS7 cells and COS7 cells transfected with vector, proving the validity of mouse Pgam1 and Pgam2 cDNAs we report here. Pgam1 and Pgam2 were assigned to 116.16 cR from D19Mit52 and 29.57 cR from D11Mit129, respectively, by radiation hybrid method. The partial genomic sequence of Pgam2 was determined, which showed that mouse Pgam2 consisted at least three exons and two introns. In addition, a pseudogene of Pgam1, Pgam1-ps1, was identified from mouse genomic sequence.

cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology withDrosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity withDrosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that humanGGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. TheGGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest thatGGPPS may be one of the candidate genes for prostate cancer.

Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovinemsrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp humanMSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine andEscherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of humanMSRA in most human tissues and with highest expression in kidney. By radiation hybrid parlel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that humanMSRA might be the candidate of the two diseases.

Application of fluorescent in situ hybridization in primary diagnosis of aplastic anemia by morphology

OBJECTIVE To investigate the clinical application of fluorescent in situ hybridization (FISH) for the differential diagnosis of myelodysplastic syndromes (MDS) and aplastic anemia (AA). METHODS A FISH kit capable of detecting the chromosomal abnormalities related to MDS was used to analyze 94 patients who were suspected to have AA by bone marrow morphology. RESULTS Cytogenetic abnormalities were detected in 11 of the 94 patients, which included trisomy 8 (5 cases), 20q- (1 case) and -Y (1 case). There were 4 cases related to MDS, which included 3 cases of 5q-, in which 1 case carry 20q- at the same time, and 7q- (1 case). No significant difference was found between the MDS and AA groups in terms of age, sex or routine blood examination including absolute neutrophil count, hemoglobin content and platelet count. CONCLUSION FISH can detect certain cytogenetic abnormalities related to MDS in patients morphologically diagnosed as AA.

Molecular cloning and tissue expression analysis of the β subunit of elongation factor 1 in the mouse.

The regulation of translation in eukaryotes might act at any one of three levels: initiation, elongation, or termination. Peptide elongation is proposed to affect rates of protein synthesis (Linz and Sypherd, 1987). There are three types of elongation factors in eukaryotes, i.e., EF-1, EF-2, and EF-3, and each has different functions. The role of EF-1 lies in the binding and transportation of aminoacyltRNAs to the ribosome; EF-1 exists in cells as a heterotetramer made up of subunits EF-1 a, -b, -g, and/or -d (Ejiri et al.,1983, Carvalhoet al.,1984, Ejiriet al., 1989). The EF-1 bg complex catalyzes the hydrolysis of GTP to GDP and the exchange of GDP for GTP on EF-1 a. The actual guanine nucleotide exchange activity resides in the EF-1 b moiety. EF-1g enhances such activity of EF-1 b. Recent research indicates that EF-1 may play important roles in cell growth and senescence, though the mechanism is not yet known (Giordano et al., 1989). Consisting of unequal quantities of EF-1 a, -1b, -1g, and/or -1d, EF-1 has several molecular forms, with molecular weights ranging from 50,000 Da to several million daltons (Leutheret al.,1993), and different forms of EF-1 have different influences on the protein synthesis rate, which is probably one of the reasons why EF-1 could affect the process of cell growth and senescence. Up till now, chicken EF-1b (GenBank No. AF103726), rabbit EF-1b (GenBank No. X74728), and two human EF-1b (GenBank Nos. X60489 and X60656) cDNAs have been cloned (Sanders t al.,1991). In this paper, a cDNA sequence was cloned from a mouse heart cDNA library. The deduced protein of

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF A NOVEL HUMAN CDNA FRAGMENT ENCODING A PUTATIVE SER/THR PROTEIN KINASE

A novel full-length cDNA encoding a putative serine/threonine kinase has been isolated from a human testis cDNA library. A nucleotide sequence of 1225 bp length has been determined containing an open reading frame of 1 044 nucleotides (encoding 348 amino acids). In view of its degree of homology to members of the Ser/Thr protein kinase family and the closest relationship toMus musculus STK-1, the predicted product was designated by the name of HUMSTK-1. Its mRNA is present in large amounts in thymus, and small amounts in testis, small intestine and colon.