Adult muscle sodium channel alpha-subunit is a gene candidate for malignant hyperthermia susceptibility.

Human myosin light chain-2 (MYL2) is an important protein involved in the regulation of myosin ATPase activity in smooth muscle. In cardiac muscle, the precise role of MYL2 is not well understood; however, an increase in ventricular MYL2 is observed during myocardial hypertrophy in cardiac patients with valve stenosis. The chromosomal location of the gene coding for MYL2 was identified using a cloned cDNA for human MYL2. Southern blot analysis of DNA from a human/rodent somatic cell hybrid mapping panel showed that the BamHI fragment that hybridized with this cDNA probe was concordant with chromosome 12. The 768-bp cDNA was hybridized to human metaphase chromosomes. The results revealed a significant clustering of silver grains over chromosome 12 bands q23-q24.3, indicating that the gene coding for MYL2 is located in this region.     

Assignment of the structural gene coding for albumin to human chromosome 4

Summary Albumin is a developmentally regulated serum protein synthesized in the liver mainly during adulthood. Family studies using variant forms of albumin established autosomal linkage between albumin and group-specific component protein (GS). Since GC has been assigned to human chromosome 4, albumin can be indirectly assigned to the same chromosome; however no direct assignment has been made. Recently, the human albumin cDNA probe has been isolated and characterized. It thus permits a direct chromosomal assignment of the albumin gene in the human genome. When the cDNA probe was hybridized to the HindIII digested total human DNA, an intense band at 6.8 kb was present. When the probe was hybridized to the HindIII digested Chinese hamster CHO-K1 DNA, a less intense band at 3.5 kb was found, plus three other faint bands. When the probe was hybridized to a series of human/CHO-K1 cell hybrids retaining a complete hamster genome and various combinations of human chromosomes, it was evident that hybrids containing human albumin gene sequences could be readily distinguished from hybrids containing no human albumin gene. Analysis of 22 primary cell hybrids for the presence or absence of human albumin sequences has assigned the albumin gene to human chromosome 4. Similar results were obtained using another restriction endonuclease EcoR1. Thus, by direct assay of the genomic albumin gene sequences in the cell hybrids, we provide evidence for a direct assignment of the structural gene for human albumin to chromosome 4.     

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum.

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.     

Regional assignment of the erythropoietin gene to human chromosome region 7pter----q22

The chromosomal location of the human gene for erythropoietin (EPO) was determined by Southern blot hybridization analysis of a panel of human-mouse somatic hybrid cell DNAs. DNAs from cell hybrids containing reduced numbers of human chromosomes were treated with the restriction enzyme PstI and screened with a cloned human EPO cDNA probe. EPO is assigned to human chromosome 7 based on the complete cosegregation of EPO with this chromosome in all 45 cell hybrids tested. A cell hybrid containing a translocated derivative of chromosome 7 localizes EPO to 7pter----q22. A HindIII restriction fragment length polymorphism is detected by hybridization of the EPO cDNA probe to human genomic DNA.     

Identification of the myelin protein plasmolipin as the cell entry receptor for Mus caroli endogenous retrovirus

The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.     

Assignment of human aldolase C gene to chromosome 17, region cen→q21.1

Summary The mapping of the gene coding for human aldolase C has been studied using a specific cDNA probe and genomic blots from a panel of human-hamster somatic cell hybrids. The results show that the aldolase C gene is on chromosome 17. In situ experiments have restricted the mapping to the region 17cenq21.1. Using the same panel of human-hamster somatic cell hybrids, we have confirmed the localization of aldolase A and B on chromosomes 16 and 9, respectively.     

The gene for the alpha polypeptide of pyruvate dehydrogenase is X-linked in humans.

The chromosomal location of the gene for the alpha polypeptide of the pyruvate dehydrogenase (alpha E1), a major component of the pyruvate dehydrogenase complex, was determined by using a cloned cDNA for alpha E1. This 1-kb cDNA was isolated from a human liver lambda gt11 expression library with specific antibodies and included the coding (from amino acid 144 to the carboxy terminus) and the 3' untranslated regions. Southern blot analysis of the DNA from a panel of rodent-human hybrid cells showed that the absence or the presence of the major EcoRI fragment that hybridized with this cDNA probe was concordant with the presence of the Xq24-p22 region of the human X chromosome. The result of in situ hybridization with human metaphase chromosomes further mapped the alpha E1 gene to the Xp arm.     

Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.1

Summary The gene for human mineralocorticoid receptor (hMR), previously mapped to chromosome 4, has been further localized to 4q31.1 by in situ hybridization using a biotinylated 3.75kb human cDNA clone encoding the primary amino acid sequence of hMR as a probe. Preliminary comparative mapping studies in orangutan (Pongo pygmaeus) suggest localization of the probe to the long arm of chromosome 3.     

The gene for clotting factor 10 is mapped to 13q32----qter

The structural gene for the human clotting factor 10 (F10) has been mapped to chromosome 13 with a cDNA probe hybridized to DNAs from a panel of human X hamster hybrids. In situ hybridization was used to assign F10 to region 13q32----qter of chromosomes from normal human lymphocytes.     

Localization of the beta A4-crystallin gene (CRYBA4) on human chromosome 22 in the region q11.2-->q13.1.

The chromosomal localization of the human gene (CRYBA4) coding for the eye lens protein beta A4-crystallin has been carried out using a nearly full-length cDNA clone encoding bovine beta A4-crystallin. A panel of 21 human-mouse or human-hamster hybrid cell lines derived from different parental combinations was characterized with respect to the human chromosomal content and the presence of well established human chromosome-specific markers. These panels were screened for the presence of CRYBA4 using the bovine cDNA clone as a probe. A 100 percent concordance was observed between the presence or absence of the CRYBA4 and human chromosome 22 indicating that the gene resides on this chromosome. By using cell hybrids containing translocated chromosome 22 segments, the localization could be refined to the region 22q11.2-->q13.1.     

The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies a TaqI RFLP.

Human dopaminergic neurons are involved in the control of hormone secretion, voluntary movement, and emotional behavior. Mediating these effects are the dopamine D1 and D2 receptors. These macromolecules belong to a large family of related sequences known as the G protein-coupled receptors. The D2 receptors have been of special interest because they bind, with high affinity and specificity, many of the commonly prescribed antipsychotic drugs. We previously isolated a full-length cDNA clone of the rat D2 receptor. When a chromosome mapping panel was probed with the rat D2 receptor cDNA a 15-kb EcoRI restriction fragment was identified and localized to human chromosome 11. The rat cDNA was also used to clone a human genomic fragment, lambda hD2G1, which contains the last coding exon of the D2 receptor gene (DRD2) and 16.5 kb of 3' flanking sequence. Hybridization of lambda hD2G1 to a chromosome 11 regional mapping panel localized DRD2 to 11q. In situ hybridization of lambda hD2G1 to metaphase chromosomes refined this assignment to the q22-q23 junction of chromosome 11. A search for RFLPs associated with D2DR identified a frequent two-allele TaqI RFLP.     

Assignment of two rat dihydrofolate (DHFR) genes to chromosomes 2 and 4

A panel of rat x mouse cell hybrids was used in the chromosomal mapping of the rat dihydrofolate reductase (DHFR) gene. It was determined that the probe hybridized to gene sequences on two different chromosomes (Nos. 2 and 4), possibly representing the active gene and a pseudogene. Hybridization of the DHFR probe to DNA from a methotrexate resistant rat cell line revealed that the gene on chromosome 2 was amplified, but not the gene on chromosome 4. This result was taken to suggest that the active DHFR gene is located on rat chromosome 2 and that the sequence on chromosome 4 is a pseudogene.     

Regional mapping of the human placental alkaline phosphatase gene (ALPP) to 2q37 by in situ hybridization

In order to determine the subchromosomal location of the gene for human placental alkaline phosphatase (ALPP; EC 3.1.3.1.), a cDNA probe encompassing most of the ALPP translated sequences was hybridized in situ to metaphase chromosomes. Our results confirm previous assignment of the gene to chromosome 2 and allow its regional mapping to band q37.     

Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.     

Chromosome 3q (22-ter) encodes the human transferrin receptor

The human transferrin receptor is an integral membrane glycoprotein of 180,000 molecular weight (mol. wt.) formed from two subunits of 90,000 mol. wt. A clone panel of Chinese hamster-human somatic cell hybrids was screened using a single cell plating cytotoxicity assay and rabbit antiserum raised to purified human transferrin receptor. Chromosome 3 displayed the highest rate of concordance with the presence of human transferrin receptor, as assayed by cytotoxicity. Antitransferrin receptor serum-resistant segregants of chromosome 3 positive, receptor-positive hybrids were selected, using antiserum and complement. The segregants consistently lost chromosome 3. 125I human transferrin binding studies confirmed synteny between the functional human transferrin receptor and chromosome 3. Examination of hybrids with either translocated or deleted chromosome 3's allows regional mapping to 3q(22-ter).     

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD)

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.     

Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.     

The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization

The Type I (mineralocorticoid) receptor has identical affinities in vitro for cortisol and aldosterone. It has been suggested that the selective role of aldosterone in regulating sodium homeostasis relies on the microsomal enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD). This enzyme converts cortisol to its inactive metabolite, cortisone, preventing cortisol from binding to the Type I receptor. We have isolated human cDNA clones encoding 11-HSD from a human testis cDNA library by hybridization with a previously isolated rat 11-HSD cDNA clone. The cDNA contains an open reading frame of 876 bases, which predicts a protein of 292 amino acids. The sequence is 77% identical at the amino acid level to rat 11-HSD cDNA. The mRNA is widely expressed, but the level of expression is highest in the liver. Hybridization of the human 11-HSD cDNA to a human-hamster hybrid cell panel localized the single corresponding HSD11 gene to chromosome 1. This gene was isolated from a chromosome 1 specific library using the cDNA as a probe. HSD11 consists of 6 exons and is at least 9 kilobases long. The data developed in this study should be applicable to the study of patients with hypertension due to apparent mineralocorticoid excess, a deficiency in 11-HSD activity.