Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.     

Chromosomal assignment of the human immunophilin FKBP-12 gene

FKBP-12 is the major T cell binding protein for the immunosuppressive drugs FK506 and rapamycin. It is a member of the immunophilin family of proteins which are believed to play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. The chromosomal assignment of the human FKBP-12 gene was determined by using the polymerase chain reaction to amplify an intron-containing region of the gene in purified DNA isolated from 42 human-rodent somatic cell hybrids. The results of this analysis indicated that the FKBP-12 gene resides on human chromosome 20.     

Organization of the human protein S genes

Human genomic clones that span the entire protein S expressed gene (PS alpha) and the 3' two-thirds of the protein S pseudogene (PS beta) have been isolated and characterized. The PS alpha gene is greater than 80 kilobases in length and contains 14 introns and 15 exons, as well as 6 repetitive "Alu" sequences. Exons I and XV contain 112 and 1139 bp 5' and 3' noncoding segments in addition to the amino and carboxyl termini, respectively. Exons I-VIII encode protein segments that are homologous to the vitamin K dependent clotting proteins and are bounded by introns whose position and type are identical with other members of this protein family. Exons IX-XV encode protein segments homologous to sex hormone binding globulin (SHBG) and are bounded by introns of identical type and position as in the SHBG gene. Genomic clones for the PS beta gene cover a distance of greater than 55 kilobases and contain segments corresponding to amino acids 46-635 of the mature protein and the 1.1-kb 3' noncoding region of the cDNA. The presence of multiple base changes in the coding portions of this gene, resulting in termination codons and frame shifts, suggests that it is a pseudogene. Comparison of DNA sequences for the two genes reveals 97% identity for coding and 3' noncoding, and 95.4% for intronic regions, suggesting divergence of the two genes is a relatively recent event.     

Structural organization of the gene for the alpha 1 chain of human type IV collagen

The complete exon size and distribution pattern in the gene for the alpha 1 chain of human type IV collagen was determined. Clones covering 145 kilobases (kb) of genomic DNA including 100 kb of the gene itself as well as 25 kb upstream and 20 kb downstream of the gene sequences, respectively, were isolated from lambda phage and cosmid libraries. The overall gene structure was determined by endonuclease restriction mapping and R-loop analyses and all exon sizes by nucleotide sequencing. The characterized clones contained all the coding sequences except for exon 2 whose sequence was determined after its amplification by the polymerase chain reaction. There were four gaps in the intron sequences; the exact size of the gene is unknown. The entire gene is at least 100 kb in size and contains 52 exons whose size distribution is completely different from that of the genes for fibrillar collagens. In the -Gly-X-Y- coding region there are three exons of 99, 90, and 45 base pairs (bp) each and two exons of 27, 36, 42, 51, 54, 63, and 84 bp each. The rest of the exons have sizes between 71 and 192 bp in the collagenous region. About one-half of the -Gly-X-Y- repeat coding exons start with the second base for the codon of glycine, whereas the other half starts (with two exceptions) with a complete glycine codon. The distribution of split versus unsplit codons is uneven in that the first 19 exons of the gene start with a complete codon. The gene contains repetitive sequences in several regions. A 185-nucleotide segment containing 40 copies of CCT flanked by poly(C) and poly(T) sequences was shown to be located adjacent to an exon. The gene has previously been shown to be located head-to-head to the alpha 2(IV) collagen gene at the distal end of the long arm of chromosome 13, such that the first exons of the two genes are separated by as little as 42 bp (P?schl, E., Pollner, R., and Kühn, K. (1988) EMBOJ. 7,2687-2695; Soininen, R., Huotari, M., Hostikka, S. L., Prockop, D. J., and Tryggvason, K. (1988) J. Biol. Chem. 263, 17217-17220). The results demonstrate that the human alpha 1(IV) collagen gene has a structure distinctly different from the genes for fibrillar collagens and also that it is considerably larger than any collagen gene characterized to date.     

Identification of an additional gene belonging to the alpha 2 adrenergic receptor family in the human genome by PCR.

We here describe the cloning of an additional gene, called alpha 2-1.8, which is similar to the previously cloned human alpha 2-adrenergic receptor located on chromosome 4. The alpha 2-1.8 gene was identified by using the polymerase chain reaction with primers specific for sequences in transmembrane regions 2 and 5 of the previously isolated human alpha 2-C4 and alpha 2-C10 adrenoceptor genes, which are localized on chromosomes 4 and 10, respectively. The new gene was identified by amplifying the 1.8 kb size fractionated region of PstI restriction cut human genomic DNA. The previously cloned alpha 2-C10 and alpha 2-C4 genes were recovered at their expected locations, 0.96 and 5.9 kb, respectively. We have identified 387 bases of the new alpha 2-1.8 gene, and its sequence is identical to the previously described alpha 2-C4 gene, but it is distinct from the alpha 2-C10 and alpha 2-C2 genes. Our results demonstrate that the alpha 2-C4 adrenergic receptor exists in more than one copy in the human genome.     

Assignment of the alpha 1-antitrypsin gene and a sequence-related gene to human chromosome 14 by molecular hybridization

alpha 1-Antitrypsin is a major plasma protease inhibitor synthesized in the liver. Genetic deficiency of this protein predisposes the affected individuals to development of infantile liver cirrhosis or chronic obstructive pulmonary emphysema. The human chromosomal alpha 1-antitrypsin gene has been cloned and shown to contain three introns in the peptide-coding region. When the cloned alpha 1-antitrypsin gene was used as a hybridization probe to analyze Eco RI-digested genomic DNA from different individuals, two distinct bands of 9.6 kilobases (kb) and 8.5 kb in length were observed in every case. Further analysis using only labeled intronic DNA as the hybridization probe has indicated that the authentic alpha 1-antitrypsin gene resides within the 9.6-kb fragment. Thus the 8.5-kb fragment must contain another gene that is closely related in sequence to the alpha 1-antitrypsin gene. Using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the alpha 1-antitrypsin gene as well as the sequence-related gene have been assigned to human chromosome 14 by Southern hybridization and synteny analysis.     

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.     

Chromosomal distribution of three members of the human natriuretic peptide receptor/guanylyl cyclase gene family

Chromosomal localization of the genes encoding three homologous human proteins, the ANPRA, ANPRB, and ANPRC cell surface receptors, was determined by polymerase chain reaction (PCR) analysis of genomic DNA from somatic cell hybrids. The ANPRA gene was assigned to 1q12----qter by intron-specific PCR. The ANPRB gene was assigned to 9p11----p22 using species-specific length variation in PCR fragments. The ANPRC gene was assigned to chromosome 5 using human-specific PCR primers identified by screening a human primer panel on parental DNA samples (shotgun primer screening). Chromosomal assignments based on PCR analysis were confirmed and the genes further sublocalized by in situ hybridization of cloned cDNA probes to human metaphase chromosomes. The ANPRA gene was sublocalized to 1q21----q22, the ANPRB gene to 9p12----p21, and the ANPRC gene to 5p13----p14.     

Localization of the gene for human simple epithelial keratin 18 to chromosome 12 using polymerase chain reaction.

Many human genes encoding keratin intermediate filament proteins are clustered on chromosomes 17 (the type I genes) and 12 (the type II genes). Some have not yet been localized, notably the genes for the primary embryonic keratins 8 and 18, normally expressed in simple epithelia: this is because the numerous pseudogenes for these keratins have made it difficult to identify the true functional gene in each case. Through the use of human-specific primers from within introns of the published gene sequence for human type I keratin 18, human genomic DNA has been specifically amplified using the polymerase chain reaction. A single reaction product was obtained. DNA from a characterized series of mouse-human somatic cell hybrid lines was tested for the presence of sequences able to initiate the chain reaction from these primers, and the presence or absence of this genomic DNA PCR product allowed us to assign a gene for human keratin 18 to chromosome 12 unambiguously. This differs from the location of other human type I keratins on chromosome 17 and may indicate the early divergence of the genes for stratifying cell keratins from that of simple, or embryonic, keratin 18.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Localization of the human procollagen alpha 1(IV) gene to chromosome 13q34 by in situ hybridization.

Type IV (alpha 1 and alpha 2 chains) appears to be the only procollagen present in basement membranes. The structure of this protein is highly divergent from the interstitial and type V procollagens as exemplified by the interruptions in the Gly-X-Y region and unprocessed amino and carboxyl noncollagenous peptides. To expand our knowledge concerning the primary sequence of type IV and to investigate the factors influencing its unique distribution, we recently isolated cDNA clones coding for part of the human alpha 1(IV) chain. To determine if the alpha 1(IV) gene was cytologically linked to other procollagen genes that have been assigned to autosomes 17, 12, 7, and 2, overlapping clones covering 2.6 kilobases (kb) of the alpha 1(IV) mRNA were used together for in situ hybridization to human metaphase chromosomes. Here, we show precise localization of alpha 1(IV) at the telomere of 13q, thereby defining a fifth chromosome that contains members of this large and surprisingly dispersed multigene family.     

Localization of the intronless gene coding for calmodulin-like protein CLP to human chromosome 10p13-ter

The functional intronless gene coding for a calmodulin-like protein (CLP) has been localized to human chromosome 10p13-ter. Chromosomal assignment was performed by Southern blot analysis of DNA from human-rodent somatic cell hybrids and amplification of a CLP gene-specific 1090-bp DNA fragment by the polymerase chain reaction (PCR) on DNA from human-hamster cell hybrids. Chromosomal sublocalization was carried out by in situ hybridization of human chromosome metaphase spreads. The CLP gene is the first member of the human calmodulin/calmodulin-like gene family to be chromosomally sublocalized. Its presence near the telomeric end of the short arm of chromosome 10 may be of significance with respect to its highly (epithelial) cell-type restricted expression in vivo and strong downregulation upon malignant transformation. The generation of a human CLP gene-specific sequence tag site specified by the two primers used for PCR should prove useful for future linkage studies.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.