Platelet-derived growth factor A chain: confirmation of localization of PDGFA to chromosome 7p22 and description of an unusual minisatellite.

The human PDGFA gene, encoding the A chain of platelet-derived growth factor, has been previously cloned and characterized, but two conflicting chromosomal localizations have been presented. To resolve this controversy, we have now performed nonisotopic in situ hybridization using new genomic PDGFA subclones and analyzed somatic cell hybrid DNAs for the presence of human PDGFA by polymerase chain reaction. The results confirm our previous assignment of PDGFA to chromosome 7p22. New sequence data from the PDGFA locus have been obtained and analyzed. An unusual minisatellite, which includes an evolutionarily conserved protein-coding region of exon 4, was found within IVS4. The minisatellite includes an embedded polymorphic pentanucleotide microsatellite repeat. Analysis of this polymorphism and in situ hybridization both locate PDGFA outside the monosomic region in a patient with a de novo deletion of the short arm of chromosome 7 [del (7)(p22.1-pter)].     

人SDCT2基因的两种不同转录产物选择性转录机理分析

为了克隆人高亲和力钠离子依赖性二羧酸转运蛋白 (highaffinitysodium dependentdicarboxylatetransporter,SDCT2 ,或NaDC3)基因并研究其生理功能 ,用大鼠SDCT2基因序列作为电子杂交探针对人EST数据库进行电子筛选 ,得到了一系列与大鼠SDCT2序列具有高度同源性的人EST序列 ,将它们拼接成 2个基因重叠群 ,设计特异性PCR引物通过RT PCR扩增得到 2条杂交探针用于筛选人肾cDNA文库 .从肾组织中同时克隆出了人SDCT2基因 2种mRNA变异体的全长cDNA(SDCT2α和SDCT2 β) ,两者 5′端前 3435bp序列完全一致 ,但 3′端长度不同 ,SDCT2 β在第 3435bp以后比SDCT2α多出了 5 85bp的序列 .Northern杂交和RT PCR显示 ,SDCT2α在人肾中的表达丰度最高 ,在肝、脾、胎盘、脑及结肠中也有低水平的表达 .而SDCT2 β主要在肾脏中表达 ,在脾也有低水平的表达 .基因组结构分析表明 ,虽然两种mRNAs均由 13个外显子组成 ,但是SDCT2α的第 13外显子含有 1个poly(A)加尾信号AATAAA ,而SDCT2 β的第 13外显子含有 2个poly(A)加尾信号 .这表明在肾脏和脾脏组织中 ,人SDCT2基因可能通过选择性使用位于第 13外显子不同位置的 2个poly(A)信号而转录出 2种不同长度的mRNA变异体 .     

The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen

The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

A simple method using β-globin polymerase chain reaction for the species identification of animal cell lines—A progress report

Continuous cell lines are widely used in cell biology and serve as model systems in basic and applied research. Fundamental requirements for the use of cell lines are a well-identified origin and the exclusion of cross-contamination by prokaryotic or eukaryotic cells. Because the cross-contamination of one cell line with another cell line may occur in a concealed manner, special emphasis must be taken to (1) prevent such an "accident" and (2) monitor regularly the identity of the cell line(s) in use. Apart from human cell lines, mouse-, rat-, and hamster-derived cell lines are used in basic cell culture and biotechnology. We established a polymerase chain reaction (PCR) assay to detect and confirm the species origin for these species and to detect interspecies cross-contamination. Our PCR method is based on oligonucleotide primers annealing to specific sequences in the beta-globin gene, which were designed to amplify one deoxyribonucleic acid (DNA) segment only per analyzed sample. We confirmed the species identity of 82 cell lines as human, mouse, rat, and Syrian hamster by beta-globin PCR. The DNAs from eight additional cell lines of less frequently used species were not amplified with the primers chosen. Cross-contamination of 5-10% of either mouse or rat DNA was detectable. One species-specific primer pair was sufficient for confirmation of the expected species, and for identification of an unknown cell line the combination of two or more primer pairs is suggested. Our PCR assay represents a powerful, fast, easy, robust, and inexpensive method for speciation and does not need any elaborate sequencing or computer-based analysis system.     

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.     

K-tuple frequency in the human genome and polymerase chain reaction.

The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.     

Identification of a conserved microsatellite site in the porcine and bovine insulin-like growth factor-I gene 5'' flank

Examination of published rat and human sequences for the insulin-like growth factor-I (IGF-I) gene indicated the presence of CA dinucleotide repeats in corresponding segments of each. Presence of similar microsatellite sequences in the porcine and bovine IGF-I genes was hypothesized. A 1200-bp segment upstream of the porcine and bovine IGF-I genes was amplified using the polymerase chain reaction (PCR) with primers developed from a consensus of human, rat and bovine sequences. Both porcine and bovine PCR products contained similar microsatellite sequences. Amplified pIGF-I DNA was cloned and sequenced, and an additional primer was developed specifically for microsatellite marker detection. Six allelic variants of 124, 130, 132, 134, 136 or 138 bp were observed in pigs with differing frequencies between breeds (P < 0.01). The same primers were used to amplify the corresponding bovine microsatellite. Three alleles of 126, 128 and 130 bp were observed in a genetically diverse cattle population with estimated frequencies of 0.06, 0.68 and 0.26, respectively. Results of this study indicate sequence information from the human and laboratory species can be used to facilitate genetic marker development in livestock species.