Chromosomal localization and racial distribution of the polymorphic human dihydrofolate reductase pseudogene (DHFRP1).

The human dihydrofolate reductase (DHFR) gene family comprises one functional gene and at least four intronless processed pseudogenes. The functional DHFR gene is on chromosome 5, and DHFRP4 is on chromosome 3. Using in situ hybridization, we have now localized the functional DHFR gene to the region q11.1-q13.3 on chromosome 5. By genomic DNA analysis of a panel of human X rodent somatic-cell hybrids, we determined the chromosomal assignment of the DHFRP1 pseudogene to chromosome 18 and that of the DHFRP2 pseudogene to chromosome 6. The DHFRP1 pseudogene exhibits a novel form of polymorphism in humans in that it is present in the DNA of some individuals and absent in that of others. We investigated the racial distribution of this pseudogene in five racial groups. The allelic frequency as defined by analysis of 180 chromosomes was found to be 94% in Mediterraneans, 77% in Asian Indians, 67% in Chinese, 57% in Southeast Asians, and 32% in American blacks. These data suggest that the transposition of this "perfect" pseudogene occurred prior to the inception of the human racial groups.     

Localization of enolases 1 and 2 on chromosomes 1 and 12 respectively by the analysis of human-mouse hybrids]

The study of enolase in man-mouse somatic hybrids confirms synteny between ENO1 and the markers on human chromosome 1 (AK2, PGM1, Pep-C) and synteny between ENO2 and the markers on human chromosome 12 (LDHB, Pep-B). The study also shows that the different enolase bands observed in mouse cell strains (Cl1D, R4, A9, 3T3), in hamster cell strains (CH, V79/4, A3), and in 3 of the different bands observed in human fibroblasts have a dimeric structure. The formation of these enolase bands depends on genes at two different loci alpha and beta. The hamster cell line CH (HGPRT) showed a rare enolase phenotype with a two-banded pattern in the intermediate region, a triple-banded pattern in the slow region, and one single isozyme in the fast region. This hamster strain is heterozygous for the first locus and homozygous for the second one. The relationship between these different enolase bands is as follow: in the slow "a" zone, alpha1alpha1,alpha1 alpha2,alpha2alpha2; in the intermediate "i" zone, alpha1beta1, alpha2beta1; and, in the fast "b" zone, beta1beta1. It appears that the frequency of heterozygotes for the alpha or beta loci in man is very low. Of 32 unrelated fibroblast strains investigated, none was found to be heterozygous for the alpha or beta locus.     

Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively

An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates a wide variety of cellular processes. The enzymatic activity and intracellular localization of PP2A are determined by three distinct families of cellular regulatory subunits (B, B', and B'). The B' subunit, also known as B56, is the most diverse, consisting of five isoforms (alpha, beta, gamma, delta, and epsilon). The gene encoding B56gamma has been designated as PPP2R5C and encodes three differentially spliced variants: B56gamma1, -gamma2, and -gamma3. However, conflicting chromosomal loci have been reported in human genomic databases. The original cytogenetic mapping placed the gene on chromosome 3p21.3, whereas subsequent studies using radiation hybrid analysis localized PPP2R5C to chromosome 14q. In this study, by radiation hybrid mapping, FISH analysis, BAC clone sequencing, and RT-PCR analysis, we show that the functional gene PPP2R5C exists at 14q32.2 and gives rise to three splicing variants, B56gamma1, -gamma2, and -gamma3, whereas a nonfunctional B56gamma1 pseudogene, PPP2R5CP, is present at 3p21.3. We also report the genomic organization of both the functional gene and the pseudogene.     

Chromosomal localization of human Na+, K+-ATPase alpha- and beta-subunit genes

Na+, K+-ATPase is a heterodimeric enzyme responsible for the active maintenance of sodium and potassium gradients across the plasma membrane. Recently, cDNAs for several tissue-specific isoforms of the larger catalytic alpha-subunit and the smaller beta-subunit have been cloned. We have hybridized rat brain and human kidney cDNA probes, as well as human genomic isoform-specific DNA fragments, to Southern filters containing panels of rodent X human somatic cell hybrid lines. The results obtained have allowed us to assign the loci for the ubiquitously expressed alpha-chain (ATP1A1) to human chromosome 1, region 1p21----cen, and for the alpha 2 isoform that predominates in neural and muscle tissues (ATP1A2) to chromosome 1, region cen----q32. A common PstI RFLP was detected with the ATP1A2 probe. The alpha 3 gene, which is expressed primarily in neural tissues (ATP1A3), was assigned to human chromosome 19. A fourth alpha gene of unknown function (alpha D) that was isolated by molecular cloning (ATP1AL1) was mapped to chromosome 13. Although evidence to date had suggested a single gene for the beta-subunit, we found hybridizing restriction fragments derived from two different human chromosomes. On the basis of knowledge of conserved linkage groups on human and murine chromosomes, we propose that the coding gene ATP 1B is located on the long arm of human chromosome 1 and that the sequence on human chromosome 4 (ATP 1BL1) is either a related gene or a pseudogene.     

Localisation of neurone-specific enolase (ENO2) to 12p13

We have localised the human cDNA for neurone-specific enolase (ENO2) to chromosome region 12p13 by in situ hybridisation. Two additional smaller peaks of hybridisation to specific chromosomal subregions were observed. That on chromosome 1p36 probably represents cross-hybridisation to the locus for nonneuronal enolase (ENO1), which has been previously localised to this chromosome and with which ENO2 shares homology. A further gene for a member of the enolase family may be responsible for the hybridisation to chromosome 17.     

X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex

The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.     

An unusual adenine phosphoribosyltransferase pseudogene is syntenic with its functional gene and is flanked by highly polymorphic DNAs.

A mouse adenine phosphoribosyltransferase (aprt) pseudogene that had previously been recovered from a BALB/c sperm DNA library possessed several unusual features. Its nucleotide sequence, like that of other processed pseudogenes, was colinear with its corresponding mRNA, but it was truncated at its 3' end and lacked a poly(A) tail. The pseudogene was 82% homologous with corresponding regions of the functional gene and had incurred mutations that included transitions, transversions, deletions, and a point insertion. Even though the pseudogene was truncated within the protein-coding region of the corresponding functional gene, it was flanked at both ends by 13-base-pair direct repeats. Curiously, the direct repeats exhibited homology to APRT mRNA at the site of pseudogene divergence. The pseudogene appeared to be common to BALB/c and A/J mice, but it was contained on a 3-kilobase EcoRI fragment in the former strain and a 4.5-kilobase EcoRI fragment in the latter. The BALB/c and apparently the A/J pseudogene both mapped to chromosome 8, which also contains the functional aprt gene. The DNA sequences immediately surrounding the pseudogene in the two strains appeared to be similar, suggesting that the BALB/c and A/J pseudogenes are allelic. However, DNA sequences more distal to the pseudogene in the two strains appeared to vary. Thus, the EcoRI polymorphism was not due to simple loss of an EcoRI site, but was more complex. The pattern of flanking restriction sites was different for each of several enzymes, consistent with extensive DNA rearrangement. Double digests of BALB/c and A/J genomic DNAs revealed complex polymorphisms on both sides of the pseudogene. The results were consistent with insertion, deletion, or other rearrangement of DNA sequences that flank the pseudogene and suggest that this region of mouse chromosome 8 may be a region active for mutation or recombination.     

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.     

An expressed beta-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed beta-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human x Chinese hamster somatic cell hybrids cleaved with Hind III or EcoRI. Probes containing the 3' untranslated regions of the expressed gene M40 and of pseudogene 21 beta were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21----6pter, the 21 beta pseudogene (TUBBP1) to chromosome 8 region 8q21----8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.