Heterogeneity of the gamma-globin gene sequences in Japanese individuals: implication of gene conversion in generation of polymorphisms

The linked fetal globin genes (the G gamma- and A gamma-globin genes) were cloned from Japanese individuals with three different haplotypes of the HindIII polymorphisms within the gamma-globin genes. Determination of nucleotide sequences of the segment spanning from IVS2 to the 3' flanking region of each gamma-globin gene revealed that nucleotide differences are located at 43 positions and a stretch of simple GT or GC sequences. Almost half of the nucleotide changes could be accounted for by gene conversion between the G gamma- and A gamma-globin genes. We found that gene conversion had created the SacI polymorphic site just downstream of the A gamma-globin coding region. Association of the SacI polymorphic site with the HindIII polymorphic site suggests that the region containing these two sites was derived from that of the linked G gamma-globin gene through a gene conversion event. The nucleotide sequences obtained here are identical to those of the Caucasoid fetal globin genes of the same haplotypes, with the exception of some sequence changes in the hot spots of mutations. These results indicate that the sequence heterogeneity of the gamma-globin genes can be classified into three major categories according to HindIII haplotypes. The possible mechanisms of generation of the heterogeneity of the gamma-globin gene sequences are discussed.     

Isolation and characterization of the porcine growth hormone gene

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.     

Genetic polymorphism of the human ICOS gene

Inducible costimulator (ICOS) is a novel receptor belonging to the same family as CD28 and CTLA4, which regulate T-lymphocyte activation in the immune response. The genes for these molecules are located adjacent to each other on Chromosome 2q33. Many autoimmune diseases have been found to be genetically linked to or associated with genetic markers near the CTLA4 gene. However, as all three genes are closely linked and have related functions, it is possible that the findings could be explained by variation in CD28 or ICOS. Few data on genetic variation in the ICOS gene are available. We sequenced the ICOS gene in 13 healthy unrelated individuals and found eight single nucleotide polymorphisms. One was located in the first intron, and the others in the untranslated region of the last exon. The allele frequencies and linkage disequilibrium were determined from a population sample of 63 Finnish individuals. The results show that the ICOS gene is polymorphic, but no variation in the coding sequence was detected, implying that the genetic linkage of this gene region to autoimmune diseases may not result from structural variation in the ICOS molecule. These polymorphisms, however, should be useful in genetic studies of this candidate gene.     

Molecular analysis of the folC gene of Pseudomonas aeruginosa

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.     

The chimpanzee GH locus: composition, organization, and evolution

In most mammals the growth hormone (GH) locus comprises a single gene expressed primarily in the anterior pituitary gland. However, in higher primates multiple duplications of the GH gene gave rise to a complex locus containing several genes. In man this locus comprises five genes, including GH-N (expressed in pituitary) and four genes expressed in the placenta, but in other species the number and organization of these genes vary. The situation in chimpanzee has been unclear, with suggestions of up to seven GH-like genes. We have re-examined the GH locus in chimpanzee and have deduced the complete sequence. The locus includes five genes apparently organized in a fashion similar to that in human, with two of these genes encoding GH-like proteins, and three encoding chorionic somatomammotropins/placental lactogens (CSHs/PLs). There are notable differences between the human and chimpanzee loci with regard to the expressed proteins, gene regulation, and gene conversion events. In particular, one human gene (hCSH-L) has changed substantially since the chimpanzee/human split, potentially becoming a pseudogene, while the corresponding chimpanzee gene (CSH-A1) has been conserved, giving a product almost identical to the adjacent CSH-A2. Chimpanzee appears to produce two CSHs, with potentially differing biological properties, whereas human produces a single CSH. The pattern of gene conversion in human has been quite different from that in chimpanzee. The region around the GH-N gene in chimpanzee is remarkably polymorphic, unlike the corresponding region in human. The results shed new light on the complex evolution of the GH locus in higher primates.     

In vitro characterization of a human calcitonin receptor gene polymorphism

Calcitonin is a 32 amino acid peptide hormone that inhibits bone resorption by stimulating calcitonin receptors (CTR) located on the surfaces of osteoclasts. A polymorphism at nucleotide 1340 of the human calcitonin receptor gene (CALCR) lies within the coding region and has the potential to change the amino acid at codon 447 from leucine to proline. In the present study, we scanned the coding region, portions of the 5'-flanking and 3'-flanking sequences, and the intron-exon boundaries of the human CALCR gene for additional polymorphisms, and determined the frequency of the codon 447 polymorphism in several ethnic groups. Because a leucine to proline change has the potential for significant structural alteration, receptor genes encoding either leucine or proline at residue 447 were transiently expressed in COS-7 cells to determine the binding and functional consequences of this polymorphism. Our complete polymorphism scan of the CALCR gene identified 11 polymorphic sites in the gene and confirmed the presence of the previously identified nucleotide T1340C (codon 447) polymorphism. Ten of the 11 polymorphisms were single nucleotide polymorphisms (SNPs). For the codon 447 polymorphism, the prevalence of the TT genotype (leucine) was 59% in Caucasians, 27% in African-Americans, 0% in Asians, and 20% in Hispanics. The presence of this SNP appears to have no statistically significant difference with the receptor's ability to bind calcitonin or signal when activated with the hormone.     

Expression studies of transfected multigene families by homologous DNA mutagenesis

A valuable approach for multigene family studies where the expression product of at least one gene member of the family is measurable is described. In such cases, the effect on gene expression of nucleotide sequence differences or mutations occurring in other members of the family or at alleles can easily be determined. This is achieved by a strategy called homologous DNA mutagenesis. It consists of the insertion of mutated regions from homologous genes into the context of the gene coding for the assayable product. Here we demonstrate the feasibility of this approach using gene members of the human growth hormone and human placental lactogen (hGH-hPL) multigene family.     

Structure and activity of regulatory elements involved in the activation of the Hoxd-11 gene during late gastrulation.

We have used reporter gene constructs to study the cis regulation of the Hoxd-11 gene (previously Hox-4.6) in transgenic mice. We identified a 5 kb regulatory unit, which was able to reproduce important aspects of the initial activation of the gene along the major body axis. The comparison of the nucleotide sequence of this DNA fragment with the corresponding avian genomic region revealed the presence of seven highly homologous stretches of DNA outside the protein coding regions. In particular, the 3' flanking region contained two such domains that are required to mediate the embryonic activation. A chimeric construct containing the two short homologous regions from the chicken gene could replace the complete murine fragment thus demonstrating that the conserved domains carry the main regulatory elements involved in this activation. The first half of this bipartite regulatory region has enhancer activity when tested with a heterologous promoter, while the second half is required to restrict the enhancer activity to the proper expression domain. These results suggest that stage- and tissue-specific cooperation between regulatory elements is required to control properly the activity of the Hoxd-11 promoter.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

Identification and characterization of coding single-nucleotide polymorphisms within a human olfactory receptor gene cluster

Single-nucleotide polymorphisms (SNPs) were studied in 15 olfactory receptor (OR) coding regions, one control region and two noncoding sequences all residing within a 412 kb OR gene cluster on human chromosome 17p13.3, as well as in other G-protein coupled receptors (GPCRs). A total of 26 SNPs were identified in ORs, 21 of which are coding SNPs (cSNPs). The mean nucleotide diversity of OR coding regions was 0.078% (ranging from 0 to 0.16%), which is about twice higher than that of other GPCRs, and similar to the nucleotide diversity levels of noncoding regions along the human genome. The high polymorphism level in the OR coding regions might be due to a weak positive selection pressure acting on the OR genes. In two cases, OR genes have been found to share the same cSNP. This could be explained by recent gene conversion events, which might be a part of a concerted evolution mechanism acting on the OR superfamily. Using the genotype data of 85 unrelated individuals in 15 SNPs, we found linkage disequilibrium (LD) between pairs of SNPs located on the centromeric part of the cluster. On the other hand, no LD was found between SNPs located on the telomeric part of the cluster, suggesting the presence of several hot-spots for recombination within this cluster. Thus, different regions of this gene cluster may have been subject to different recombination rates.