Two human relaxin genes are on chromosome 9.

We have recently cloned two different human relaxin gene sequences. One of these (H1) was isolated from a human genomic clone bank and the other (H2) from a cDNA library prepared from human pregnant ovarian tissue. Southern gel analysis of the relaxin genes within the genomes of several unrelated individuals showed that all genomes contained both relaxin genes. Hence it is unlikely (p less than 0.001) that the two relaxin gene sequences are alleles. Rather, it is probable that there are two relaxin genes within the human genome. It is likely that relaxin and insulin genes have evolved from a common ancestral gene by gene duplication, since structural similarities between insulin and relaxin are evident at both the peptide and gene level. To investigate the evolutionary relationship between the two human relaxin genes and the insulin gene, we have determined the chromosomal position of the relaxin genes using mouse/human cell hybrids. We found that the human insulin and relaxin genes are on different chromosomes. Both human relaxin genes are located on the short arm region of chromosome 9.     

Two genes encoding distinct cytosolic glutamine synthetases are closely linked in the pine genome

The major isoenzyme of glutamine synthetase found in leaves of angiosperms is the chloroplastic form. However, pine seedlings contain two cytosolic glutamine synthetases in green cotyledons: GS1a, the predominant isoform, and GS1b, a minor enzyme whose relative amount is increased following phosphinotricin treatment. We have cloned a GS1b cDNA, and comparison with the previously reported GS1a cDNA sequence indicated that they correspond to separate cytosolic GS genes encoding distinct protein products. Phylogenetic analysis showed that the newly reported sequence is closer to cytosolic angiosperm GS than to GS1a, suggesting therefore that GS1a could be a divergent gymnospermous GS1 gene. Gene mapping using a F2 family of maritime pine showed co-localization of both GS genes on group 2 of the genetic linkage map. This result supports the proposed origin of different members of the GS1 family by adjacent gene duplication. The implications for gymnosperm genome organization are discussed.     

Two type II keratin genes are localized on human chromosome 12

Summary Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3 noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.     

Two linked genes for outer membrane proteins are absent in four non-disease strains of Haemophilus somnus

Linked genes encoding two outer membrane proteins (p76 and a family of proteins, p120) of the bovine pathogen, Haemophilus somnus, were investigated. The p120 group was previously shown to have immunoglobulin-binding activity and to react with polyclonal antiserum specific for a 270 kDa antigen (p270) which also had immunoglobulin Fc-binding activity. By Western blotting we showed that the p76 antigen also reacted with this antiserum. The p270, p120, and p76 antigens were undetectable in four serum-sensitive isolates from asymptomatic carriers but were present in the two serum-resistant virulent strains tested. Genes for p120 and p76 were subcloned on non-overlapping pUC plasmids from a cosmid (pHS1) originally cloned from a serum-resistant strain. In Escherichia coli, plasmid pHS138 expressed p76, while the p120 antigens were produced by pHS140. Southern blots of DNA from the above six strains of H. somnus using probes derived from pHS1 subclones showed that a 13.4 kb sequence was missing from the four serum-sensitive strains, but not the two serum-resistant strains. This segment included most of the insert in pHS138 and all of the pHS140 insert. The data indicate that p76 and the p120 proteins are absent from serum-sensitive strains because the coding sequences are missing, raising the possibility of insertion of these genes into the chromosome of both serum-resistant strains, or deletion from the four serum-sensitive strains.     

Tissue- and species-specific promoter elements of rat gamma-crystallin genes.

The 5' flanking regions of the six rat gamma-crystallin genes (gamma A-gamma F) are all capable of conferring lens-specific expression to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in either transdifferentiating chicken neural retina cells or mouse lens epithelial cells. Deletion mapping of the most active gamma-crystallin promoter region, the gamma D region, showed that at least three elements are required for maximal expression in mouse lens epithelial cells: element(s) located between -200 and -106, a conserved CG rich region around position -75, and a CG stretch around -15. The region between -200 and -106 was dispensable in transdifferentiating chicken neural retina cells, which instead required the region between -106 and -78. The maximal activity of the gamma E and gamma F promoters was also dependent upon the integrity of the conserved CG region located around -75. A synthetic oligonucleotide containing this sequence was capable of lens-specific enhancement of the activity of the tk promoter in transdifferentiating chicken neural retina cells but not in mouse lens epithelial cells. Our results further show that this region may contain a silencer element, active in non-lens tissues, as well.     

Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum.

Four genes immediately downstream of luxG in the Photobacterium phosphoreum lux operon (ribEBHA) have been sequenced and shown to be involved in riboflavin synthesis. Sequence analyses and complementation of Escherichia coli riboflavin auxotrophs showed that the gene products of ribB and ribA are 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthetase and GTP cyclohydrolase II, respectively. By expression of P. phosphoreum ribE in E. coli using the bacteriophage T7 promoter-RNA polymerase system, ribE was shown to code for riboflavin synthetase, which catalyzes the conversion of lumazine to riboflavin. Increased thermal stability of RibE on expression with RibH indicated that ribH coded for lumazine synthetase. The organization of the rib genes in P. phosphoreum is quite distinct, with ribB and ribA being linked but separated by ribH, whereas in E. coli, they are unlinked and in Bacillus subtilis, RibB and RibA functions are coded by a single gene.     

The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes.

Three open reading frames (ORFs) have been found in the region downstream of the luxG gene in the Photobacterium leiognathi lux operon. These genes (ORF I, II, and III) are not only closely linked to the lux operon and transcribed in the same direction but also show the same organization and code for proteins homologous in sequence to the gene products of ribB, ribA, and ribH of Bacillus subtilis, respectively. The Photobacterium leiognathi gene (ORF II) corresponding to ribA was expressed in Escherichia coli in the bacteriophage T7 promoter-RNA polymerase system and a 40 kDa 35S-labeled polypeptide has been detected on SDS-PAGE. Expression of DNA extending from luxBEG to ORF II inserted between a strong promoter and a reporter gene and transferred by conjugation into Vibrio harveyi did not affect the expression of the reporter gene. The results provide evidence that neither promoter nor terminator sites were present in the DNA between the luxG and ORF II indicating that these genes might be part of the lux operon.     

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.     

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.     

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale. Received: 19 September 1996 / Accepted: 21 May 1997     

Hsp70 genes are linked to the Xenopus major histocompatibility complex

Some of the inducible forms of the heat shock protein 70 (Hsp70) gene family are encoded in the class III region of the major histocompatibility complex (MHC) of mammals. This study was undertaken to determine whether Hsp 70 genes are linked to the MHC of Xenopus, an amphibian last sharing a common ancestor with mammals 300–350 million years ago. Segregation analyses involving seven haplotypes demonstrated the linkage of two or three inducible Hsp70 genes to the frog MHC. Another Hsp70 gene is not closely linked to the MHC. We conclude that the physical association of MHC class I and class II genes with Hsp70 genes is ancient. Correspondence to: M. F. Flajnik.     

Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes.

DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization.