Parathyroid hormone gene analysis in autosomal hypoparathyroidism using an intragenic tetranucleotide (AAAT) n polymorphism

We have identified a polymorphic tetranucleotide consisting of (AAAT) _n within the first intron of the parathyroid hormone (PTH) gene, and have used this to investigate the segregation of the PTH gene and idiopathic hypoparathyroidism in 7 affected and 21 unaffected members from three families. An association between the PTH locus and autosomal dominant idiopathic hypoparathyroidism in one family was excluded by observing recombination between the two loci. In the remaining two families with autosomal recessive idiopathic hypoparathyroidism, the PTH locus was not similarly excluded. We had previously demonstrated a donor splice site mutation of the PTH gene in one of these families, and PTH gene abnormalities were therefore sought in the second of these families. DNA sequence analysis of the three exons, together with 4 exon-intron boundaries and the promoter region of the PTH gene revealed no abnormalities, thereby indicating molecular pathology at another locus. Thus, our analysis of idiopathic hypoparathyroidism reveals genetic heterogeneity for this disorder. In addition, our indentification of a microsatellite polymorphism of the PTH gene should help further segregation studies of this locus in families with parathyroid disorders.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Characterization of two novel polymorphisms at the human parathyroid hormone gene locus

Summary Two new polymorphisms within the human parathyroid hormone (PTH) gene are described. One corresponds to a CA transversion that destroys DraII and NlaIV restriction sites. The other is revealed by the enzyme XmnI, and its position has been mapped with respect to the PTH gene. We have also identified a sequence change that results in the TaqI restriction fragment length polymorphism (RFLP) described previously at this locus and have found that this sequence change also results in disruption of a BstBI site. Finally, we describe a polymerase chain reaction (PCR)-based method that permits a rapid evaluation of the DraII and BstBI (TaqI) polymorphisms. The introduction of these two additional RFLPs and this PCR-based assay should considerably extend the power of genetic analyses of the human PTH gene.     

Nucleotide sequencing analysis of the swine 433-kb genomic segment located between the non-classical and classical<Emphasis Type="Italic"> SLA</Emphasis> class I gene clusters

Genome analysis of the swine leukocyte antigen (SLA) region is needed to obtain information on the MHC genomic sequence similarities and differences between the swine and human, given the possible use of swine organs for xenotransplantation. Here, the genomic sequences of a 433-kb segment located between the non-classical and classical SLA class I gene clusters were determined and analyzed for gene organization and contents of repetitive sequences. The genomic organization and diversity of this swine non-class I gene region was compared with the orthologous region of the human leukocyte antigen (HLA) complex. The length of the fully sequenced SLA genomic segment was 433 kb compared with 595 kb in the corresponding HLA class I region. This 162-kb difference in size between the swine and human genomic segments can be explained by indel activity, and the greater variety and density of repetitive sequences within the human MHC. Twenty-one swine genes with strong sequence similarity to the corresponding human genes were identified, with the gene order from the centromere to telomere of HCR - SPR1 - SEEK1 - CDSN - STG - DPCR1 - KIAA1885 - TFIIH - DDR - IER3 - FLOT1 - TUBB - KIAA0170 - NRM - KIAA1949 - DDX16 - FLJ13158 - MRPS18B - FB19 - ABCFI - CAT56. The human SEEK1 and DPCR1 genes are pseudogenes in swine. We conclude that the swine non-class I gene region that we have sequenced is highly conserved and therefore homologous to the corresponding region located between the HLA-C and HLA-E genes in the human.The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank databases under accession numbers AB113354, AB113355, AB113356, AB113357     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

The First Intron of Human c-fms Proto-oncogene Contains a Processed Pseudogene (RPL7P) for Ribosomal Protein L7

During sequence analysis of the first intron of the human c-fms oncogene, we identified an open reading frame encoding the ribosomal protein L7 (RPL7). The presence of this sequence within intron 1 of the c-_fms gene was confirmed by Southern blot hybridization and by sequence analysis of two independent cosmid clones (cos2-e and cos1-22) that span the human genomic c-_fms locus. The RPL7 sequence was detected in a region of sequence overlapped by the cos2-e and cos1-22 cosmid clones but oriented opposite to the c-fms gene. We demonstrated that the sequence is identical to the full-length RPL7 cDNA sequence, but lacks any recognizable introns, has a 30-bp poly(A) tail, and is bracketed by two perfect direct repeats of 14 bp. We also showed that despite the fact that the 5′ flanking region of the RPL7 sequence contains a potential TATA box upstream of an intact open reading frame, this pseudogene (RPL7P) is not actively transcribed.     

Generation of Human PTH1R Construct with Flag Epitope Located Internally: Comparison of Two-Fragment Assembly by Using PCR Overlap Extension or Ligase

Parathyroid hormone (PTH) regulates bone remodeling and calcium and phosphate homeostasis. PTH actions are mediated by type I PTH/PTH-related peptide receptor (PTH1R). There has been no commercially available, specific antibody to detect human PTH1R expression so far. Flag-tagged human PTH1R construct, converting the sequence DKEAPTGS (residues 94–101) in the exon E2 region of PTH1R to DYKDDDDK of Flag epitope, was generated by using PCR overlap extension or ligase enzyme for two-fragment assembly. We found that Flag-tagged PTH1R assembled by ligase was easy to be manipulated, but its efficiency was lower than that of PCR overlap extension. The PTH1R plasmids generated by both techniques were expressed successfully in vitro and in vivo and possessed the same physiological function as wild-type PTH1R. The Flag-tagged PTH1R construct will provide invaluable tools for study of PTH1R signaling and trafficking.     

Internal ribosome-binding site directs expression of parathyroid hormone analogue (8-84) in Escherichia coli

Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.     

The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence

The complete nucleotide sequence of the 3877-bp segment spanning the 3′ region of intron-6 to the 5′ region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exon gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40–55-million-year-old Alu-Se subclass.     

Structure and Methylation-Based Silencing of a Gene (DBCCR1) within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32–q33

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32–q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designatedDBC1(for deleted in bladder cancer gene 1). We have identified a novel gene,DBCCR1,in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entireDBCCR1cDNA sequence identified several human ESTs and a few homologous mouse ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. AlthoughDBCCR1was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5′ region of the gene and the induction ofde novoexpression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings makeDBCCR1a good candidate forDBC1.