Chromosome mapping of the human genes encoding the MAP kinase kinase MEK1 (MAP2K1) to 15q21 and MEK2 (MAP2K2) to 7q32

Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence. In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases. We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2. Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21. We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21. The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31

Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

Human homologs of two testes-expressed loci on mouse chromosome 17 map to opposite arms of chromosome 6

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.     

Assignment of the gene coding for hepatocyte growth factor-like protein to mouse chromosome 9

The gene coding for hepatocyte growth factor-like protein has been localized to mouse chromosome 9 at a locus (Hgfl) distal to the Trf locus. The likely gene order in this region is centromere-Trf-Gnai-2-Hgfl-Cck. The region surrounding the Hgfl locus shows homology of syntenty to band p21 on human chromosome 3.     

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.     

Chromosomal localization of the gene for a human thyroid hormone-binding protein.

A cDNA for the gene that encodes for a human cellular thyroid hormone-binding protein (p55) has recently been isolated and sequenced. The sequence of p55 indicates that it is identical to the protein disulfide isomerase and the beta-subunit of prolyl 4-hydroxylase. By in situ hybridization, the gene for p55 was localized on chromosome 17 at band q25. This localization shows that the p55 gene is not linked to either erbA1 or erbA2; two other thyroid hormone-binding protein genes are located at 17q 11-21 and 3p21-pter, respectively. The localization of p55 gene will permit the evaluation of the possible effects of chromosome changes on the structure and activity of the p55 gene in chromosome syndromes or neoplasms.     

The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17

The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype.     

Chromosome localization of human ARH genes, a ras-related gene family

The human ARH genes (previously called RHO) share several properties with the ras gene family. Three members of the ARH family, the H6, H9, and H12 genes, have been localized to human chromosomes 2, 5, and 3, respectively. Analysis of DNAs from a rodent-human somatic cell hybrid panel demonstrates linkage of H6 to chromosome region 2p12----2pter and H9 to region 5q33----5qter. In situ chromosome hybridization also showed that the primary site for H9 is in the 5q31----qter region. The H12 gene was some-what difficult to localize using rodent-human hybrids because the probe detects a family of rodent genes as homologous to the human probe as in the human cognate gene. However, chromosome in situ hybridization revealed grains clustered in region 3p14----3p22 with a significant peak in band 3p21. We conclude that H6 is in 2p12----pter, H9 in 5q31----5qter, and H12 in 3p21.     

The T cell receptor beta chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.     

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.     

Purification and crystallization of cyclin-dependent kinase inhibitor p21.

p21, a universal inhibitor of mammalian cyclin-dependent kinases (CDK), regulates cell cycle progression by forming various distinct protein complexes with cyclins, CDKs, and the proliferating cell nuclear antigen. We have overexpressed recombinant human p21 in E. coli and purified active p21 to near homogeneity on a large scale. Crystals of recombinant p21 have been grown in the space group P2(1) a = 157.4, b = 152.7, c = 90.6 A, and beta = 92.7 degrees. The diffraction data of the recombinant p21 have been collected to 2.5 and 3.5 A resolution for the native crystal and two heavy atom derivatives of mercury and iridium.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.     

Structure of genes encoding steroidogenic enzymes

Synthesis of adrenal steroid hormones from cholesterol entails the actions of only five enzymes, four of which are specific forms of cytochrome P450. These cytochrome P450 enzymes have all been isolated and their activities reconstituted in vitro, showing that each enzyme catalyses multiple steroidal conversions. Genes or complementary DNAs have been cloned for human P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase). The sequences for microsomal P450c17 and P450c21 are much more closely related to one another than either is to the sequence for mitochondrial P450scc. Each of these P450 enzymes is encoded by a single human gene; the gene for P450scc lies on chromosome 15, that for P450c17 lies on chromosome 10, and that for P450c21 lies on chromosome 6. The human, mouse and bovine genomes each have two P450c21 genes. While only one of these is active in mouse and man, both genes may be active in cattle. A wide variety of lesions in the human P450c21(B) gene causes congenital adrenal hyperplasia, a common genetic disorder.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

The ARF-p53 senescence pathway in mouse and human cells

Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF-p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.