Characterization of two nonsense mutations in the human dystrophin gene.

Forty Duchenne muscular dystrophy patients from the province of Moravia in the Czech Republic, who were previously found negative for large deletions in the dystrophin gene, were tested for the presence of point mutations in selected exons. Besides several intron and exon polymorphisms, two cases of nonsense mutations were detected in exon 70, thus causing the loss of the C-terminal domain of dystrophin. One of these, the mutation, S3365X, is newly reported here while the other, R3381X, has been described previously. These mutations, only 16 bp distant from each other, have a very different impact on the mental abilities of the corresponding patients.     

Characterization of mouse lines transgenic with the human poliovirus receptor gene

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.     

Characterization of human TMEM16G gene in silico

TMEM16A, TMEM16B, TMEM16C, TMEM16D, TMEM16E, TMEM16F and TP53I5 are TMEM16 family eight-transmembrane proteins with N- and C-terminal tails facing the cytoplasm. TMEM16A gene at human chromosome 11q13.3 is amplified in head and neck tumors, and TMEM16E gene at human chromosome 11p14.3 is mutated in gnathodiaphyseal dysplasia (GDD). Ngep cDNA (NM_207031.1) is derived from mouse Tmem16g gene. Here, we characterized human TMEM16G gene by using bioinformatics. TMEM16G gene, consisting of 25 exons, was located at human chromosome 2q37.3. Intra-species comparative genomics revealed that the PASK-PPP1R7-TMEM16G-HDLBP-NEDD5 locus was the unique region without paralogous region. TMEM16G mRNA was preferentially expressed in normal prostate and prostate cancer. Complete coding sequence of TMEM16G cDNA was determined by assembling 25 exons of TMEM16G gene. Human TMEM16G gene was found to encode 932-amino-acid TMEM16G protein with TM16H1, TM16H2 and TM16H3 domains. Comparative proteomics revealed that T844N amino-acid substitution occurred in human TMEM16G during evolution. TMHMM2 program predicted that mouse Tmem16g and artificial human TMEM16G (844T) were eight-transmembrane proteins, but that wild-type human TMEM16G (844N) was a seven-transmembrane protein. These facts indicate that amino-acid substitution at codon 844 of human TMEM16G resulted in the mis-folding of the eighth transmembrane helix. Human TMEM16G with altered membrane topology might show functional divergence compared with other members of the TMEM16 family.     

Characterization of the human peroxisome proliferator activated receptor delta gene and its expression

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism. Three different PPARs; alpha (PPARA), gamma (PPARG) and delta (PPARD) have been characterized and they are distinguished from each other by tissue distribution and cell activation. In this study, the structure and detailed chromosomal localization of the human PPARD gene was determined. Three genomic clones containing the PPARD gene was isolated from a human P1 library. The gene spans approximately 85 kb of DNA and consists of 9 exons and 8 introns with exons ranging in size from 84 bp to 2.3 kb and introns ranging from 180 bp to 50 kb. All splice acceptor and donor sites conform to the consensus sequences including the AG-GT motif. Although PPARD lacks a TATA box, the gene is transcribed from a unique start site located 380 bp upstream of the ATG initiation codon. The 5' and 3' ends were mapped by rapid amplification of cDNA ends and the mRNA size of PPARD based upon the structure of the gene is 3803 bp. In addition, the chromosomal sublocalization of PPARD was determined by radiation hybrid mapping. The PPARD gene is located at 14 cR from the colipase gene and 15 cR from the serine kinase gene at chromosomal region 6p21.2.     

Characterization and localization to human chromosome 1 of human fast-twitch skeletal muscle calsequestrin gene

A genomic clone encoding human fast-twitch skeletal muscle calsequestrin was isolated, and the amino acid sequence of the protein and the exon-intron boundaries of the gene were deduced from its sequence. A comparison with the rabbit gene showed that the sequence Glu-Asp-Asp-Asp-Asp near the COOH terminus of the rabbit sequence is lacking in the human gene. The calsequestrin gene was assigned to human chromosome 1 through the use of a human-mouse somatic cell hybrid mapping panel.     

Characterization of FMN2 gene at human chromosome 1q43

Mouse Formin (Fmn1) is an actin regulator interacting with Profilin, SRC, EMS1, FNBP1, FNBP2, FNBP3, FNBP4, WBP4 and alpha-catenin. FMN1, FHOD1, FHOD3, GRID2IP and FHDC1 are non-FDD-type Formin homology proteins, while FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2 are FDD-type Formin homology proteins. Here, we characterized human FMN2 gene by using bioinformatics. Complete coding sequence of human FMN2 cDNA was determined by assembling AL359918, AL513342, AL590490, AL646016 genome sequences, AF218941 partial cDNA, and AF218942 partial cDNA. FMN2 mRNA was expressed in fetal brain, adult whole brain, hypothalamus, retina, pancreatic islet and germinal-center B cells. Among various human tumors, FMN2 mRNA was expressed in parathyloid tumor, glioblastoma, retinoblastoma and chondrosarcoma. Human FMN2 (1722 aa) showed 74.7% total-amino-acid identity with mouse Fmn2, and 31.9% total-amino-acid identity with human FMN1. Although N-terminal half was divergent between FMN2 orthologs and FMN1 orthologs, FH1 and FH2 domains were conserved among FMN2 and FMN1 orthologs. Exon-intron structure was conserved between FMN2 and FMN1 genes. RYR2-FMN2-CKTSF1B2 (PRDC) locus at human chromosome 1q43 and RYR3-FMN1-CKTSF1B1 (Gremlin) locus at human chromosome 15q13-q14 were paralogous regions (paralogons) within the human genome. This is the first report on comprehensive characterization of the human FMN2 gene.     

Characterization of the human homologue of the mouse Tg737 candidate polycystic kidney disease gene

We previously identified a gene from the mutant locus in a newmouse mutation that causes recessive polycystic kidney disease.Here we describe the cloning, characterization and mapping ofthe homologous human gene. The human and mouse genes are 95%identical at the predicted amino acid sequence level, and bothgenes encode a putative protein that contains a tetratricopeptiderepeat motif. The human gene, called hTg737, is expressed witha broad tissue distribution that includes the kidney and liver,and gives rise to a 2.9 kb mRNA. The gene contains 26 exonsand spans a genomic region greater than 100 kb. Chromosome mappingexperiments revealed that the hTg737 gene maps near the centromereon the long arm of human chromosome 13, at position 13q12.1.While this gene does not map to the primary locus that has beenidentified for ARPKD in humans, it may represent a candidategene for other recessive renal disorders that have yet to bemapped.     

Characterization of the human cytomegalovirus UL75 (glycoprotein H) late gene promoter

Glycoprotein H (gH, UL75) of human cytomegalovirus (HCMV) is an essential envelope glycoprotein that functions in viral entry and the activation of gene expression. To understand the regulation of this important viral gene, the promoter of the UL75 late gene was characterized in HCMV-infected cells at the late stages of viral infection. Primer extension analysis revealed a single major start site located 26 bp downstream of a putative TATA element. Deletion analysis showed the presence of a dominant activation domain from +14 to +35 that masked regulatory sequences upstream of the TATA element. Mutational analysis demonstrated that a PEA3-like element in this downstream domain was important for promoter activation. In addition, gel shift analysis revealed direct protein binding to the PEA3-like element. Together, these studies reveal that the gH promoter is regulated in a complex manner with sequences both upstream and downstream of the cap site influencing promoter activation.     

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

Summary. The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy. Received September 2, 1997 Accepted January 14, 1998     

Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase

Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted -119 to -116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in D-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the -165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the -165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix-loop-helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3(')E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.