Genomic cloning and localization by FISH and linkage analysis of the human gene encoding the primary subunit NMDAR1 (GRIN1) of the NMDA receptor channel

A cDNA clone of the NMDAR1 (isoform E) has been used to screen both lambda and eosmid genomic libraries. A genomic phage clone was identified and sequenced and was found to contain some of the 3′ coding regions of the GRIN1 gene. This clone was used to localize the gene using fluorescent in situ hybridization (FISH) to normal chromosomes, and also to a lymphoblastoid cell line containing a translocation involving chromosomes 9 and 15. FISH localized the gene to chromosome 9q34.3. The clone was used to screen a panel of genomic DNAs cut with 20 restriction enzymes. A VNTR sequence 5′ to the gene, which was polymorphic for a number of restriction enzymes, was detected. A PvuII fragment of the genomic clone was found to detect the VNTR on Southern hybridization. The polymorphic VNTR marker was mapped against chromosome 9q34 markers using linkage analysis in the CEPH families. The GRIN1 gene was linked to D9S7 with a maximum lod score of 20·09 at zero recombination fraction in males and 0·03 % recombination in females.     

The gene for the α2 chain of the human fibrillar collagen type XI (COL11A2) assigned to the short arm of chromosome 6

A cosmid clone (CosHcol.11) containing the α2(XI) collagen gene ( COL11A2 ) has been isolated. The gene contains conserved DNA and amino-acid sequences characteristic of fibril forming collagen, which is in accordance with the classification of type XI collagen as a fibrillar collagen. The genomic clone containing the α2(XI) gene has been used as probe in the Southern blot analysis of DNA from a panel of human/hamster somatic cell hybrids containing different numbers and combinations of human chromosomes. Synteny analysis revealed that only chromosome 6 showed complete concordant segregation with C0L11A2. Furthermore, the gene was regionally mapped to the short arm of chromosome 6 by using a hybrid which contained only the long arm of the chromosome.     

Isolation and characterization of an alpha-tubulin gene from Leishmania enriettii

An alpha-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous alpha-tubulin gene from Drosophila melanogaster. A clone containing this gene has been isolated from a plasmid library of size-selected L. enriettii DNA. It was identified by hybridization with the D. melanogaster tubulin gene. The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis. The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the alpha-tubulin gene.     

The human type II collagen gene (COL2A1) assigned to 12q14.3

A cosmid clone containing the entire human type II α1 collagen gene ( COL2A1 ) was used as probe in the Southern analysis of DNA from a panel of human/hamster somatic cell hybrids containing different portions of human chromosome 12. Two of the hybrids exhibited a similar terminal deletion q14.3→qter, but one was positive for the gene while the other was negative. Therefore, the gene must reside in the region q14.3.     

Fast-twitch and slow-twitch/cardiac Ca2+ ATPase genes map to human chromosomes 16 and 12

The fast-twitch and slow-twitch/cardiac Ca ²⁺ ATPase genes have been assigned to human chromosomes 16 and 12, respectively, using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A rabbit cDNA for the fast-twitch ATPase hybridizes to a prominent single fragment in human genomic DNA digested with the restriction enzyme BamHI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosome content of the hybrids, the fast-twitch ATPase gene can be assigned to human chromosome 16. A slow-twitch/cardiac ATPase cDNA clone was isolated from a human muscle cDNA library and used to detect human fragments in EcoRI-digested somatic cell hybrid DNA. By correlating the presence of these fragments with the human chromosome content of the hybrids, the slow-twitch/cardiac ATPase gene can be assigned to human chromosome 12. Thus, the two ATPase genes, which are probably related to each other by an ancient duplication event, are not syntenic in the human genome.     

Molecular cloning and chromosomal localization of a gene coding for human cardiac myosin heavy-chain

A human cardiac myosin heavy-chain (MHC) gene, cloned in a charon 4A phage, was isolated using two rat cardiac pCMHC DNA clones (pCMHC26: alpha-MHC type; and pCMHC5: beta-MHC type) as probes and shown to correspond to cardiac myosin heavy-chain of the alpha-type. The 4.3-KB cardiac genomic DNA clone was used as a probe in the Southern analysis of human genomic DNA from human-Chinese hamster or human-mouse somatic cell hybrids. The results show that the human cardiac MHC gene is assigned to chromosome 14 and the human cardiac and skeletal MHC genes do not cosegregate as do the mouse cardiac and skeletal MHC genes.     

Parasexual analysis of human pepsinogen molecular heterogeneity

Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. Highly polymorphic variation of these proteins has been demonstrated in several populations, and comparison of the DNA restriction fragment patterns obtained from informative pepsinogen phenotypes suggest that the polymorphism results from chromosomal haplotypes containing variable numbers of pepsinogen genes. In order to isolate the three most common PGA haplotypes (A, B, and C) and to unambiguously demonstrate their relationship to the observed protein heterogeneity, we constructed mouse × human somatic cell hybrids from individuals heterozygous for PGA and INS (insulin). Here, we describe analysis of hybrid cell lines that segregated human chromosomes containing the PGAgenes and thereby provided for the parasexual discrimination of the different haplotypes on chromosome 11 determining the corresponding heterozygous phenotypes. These studies demonstrate that the A, B, and C haplotypes contain three, two, and one PGAgenes, respectively. This unusual polymorphism of genomic DNA encoding very similar proteins probably reflects recent evolution by gene duplication.     

Application of synthetic DNA probes of human alpha satellite consensus monomer for detection of centromereinvolved chromosome abnormalities

We have synthesized the alphoid monomer of 171 bp based on the consensus sequence of human alpha satellite DNA and constructed a clone of dimeric or tetrameric sequence unit. Southern blot analysis using the clone as a probe showed restriction site periodicities in human DNA digested byEcoRI orBamHI. The synthetic consensus unit could detect the alpha repeated centromeric regions of all human chromosomes by fluorescencein situ hybridization. Using the cells having a dicentric X chromosome, we showed that the two centromeric regions were stained with fluorescent alpha satellite DNA probes. Thus the probe would be useful to detect chromosomal abnormalities such as dicentrics.     

Transformation associated p53 protein is encoded by a gene on human chromosome 17

The human gene for the transformation-associated p53 phosphoprotein (P53)was assigned to the short arm of chromosome 17 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to radiolabeled DNA from a genomic clone which contained P53nucleotide sequences. Hybridization of the probe to a 2.5-kb human DNA fragment in HindIII-digested DNA was used to identify the human P53gene.     

Homologous transformation of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Summary A clone containing the Phanerochaete chrysosporium ade1 gene was isolated from a EMBL3 genomic library using the ade5 gene encoding aminoimidazole ribonucleotide synthetase, from Schizophyllum commune, as a probe. A 6.0 kb fragment incorporating the ade1 gene was subcloned into pUC18 (pADE1) and used to transform the P. chrysosporium ade1 auxotrophic strain. Transformation frequencies were similar to those obtained previously with the S. commune ade5 gene; however, homologous transformants arose earlier than heterologous transformants. The transformants were mitotically and meiotically stable and Southern blot analysis indicated that the plasmid, pADE1, integrated ectopically in single or multiple copies. The pADE1 insert was mapped for restriction sites and the approximate location of the ade1 gene within the insert was determined.     

Genomic cloning and partial characterization of human chymotrypsinogen gene

Summary Chymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescencein situ hybridization and G-banding analysis.     

Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferredoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.     

Organization of the HPRT gene and related sequences in the human genome

Comparative Southern hybridization of cDNA probes to DNA from cells carrying either one or four X chromosomes has been used to distinguish sequences derived from the functional locus for hypoxanthine-guanine phosphoribosyltransferase (HPRT) on the X chromosome from four independent HPRT-like autosomal sequences in the human genome. Subfragments of cDNA were then used to orient fragments from the HPRTlocus with respect to the mRNA sequence. The chromosomal origin of each of the autosomal sequences was determined by Southern analysis using DNA from a panel of human-Chinese hamster somatic cell hybrids. Two of the HPRT-like sequences were localized to chromosome 11, the third to chromosome 3, and the fourth to the region between p13 and q11 on chromosome 5. Three of these four autosomal sequences were isolated from genomic recombinant libraries and subcloned fragments from each were used as probes to study restriction fragment length polymorphisms (RFLP) at these loci. A RFLP for MspIwas found at the HPRT-like locus on chromosome 5 with a 1.3-kb major allele (frequency=0.8) and a 3.6-kb minor allele (frequency=0.2).     

Cloning of the orotidine 5′-phosphate decarboxylase (ODC) gene of Schwanniomyces occidentalis by complementation of the ura3 mutation in S. cerevisiae

Summary A DNA fragment containing the gene encoding orotidine 5-phosphate decarboxylase (ODC) from the yeast, Schwanniomyces occidentalis (formerly castellii) has been isolated from a genomic library constructed in the S. cerevisiae expression vector, pYcDE8. A recombinant plasmid, p2-lA, containing a 2.47 kb insert was shown to complement the ura3-52 mutation of several strains of S. cerevisiae. This DNA insert was shown to be from Schwanniomyces occidentalis by Southern hybridization analysis. A restriction enzyme cleavage map of the insert has been derived and the ODC gene localized to a 1.1 kb region by deletion analysis. In addition, we have demonstrated that expression of ODC is not dependent on the ADHI promoter carried on pYcDE8. This is the first report of the cloning of a gene from a member of the genus Schwanniomyces.     

Sequence polymorphisms of the Der p 3 house dust mite allergen

Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist. Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed. Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.     

A tandemly repetitive,centromeric DNA sequence from the Canadian woodland caribou (Rangifer tarandus caribou): Its conservation and evolution in several deer species

A highly repetitive DNA clone, designated Rt-Pst3, was isolated from thePstI digest of Canadian woodland caribou (Rangifer tarandus caribou; 2n = 70) genomic DNA. It was found to be a 991 bp monomer of a tandemly repeated DNA sequence comprising about 5.7% of the genome and localized to the centromeric regions of all caribou acrocentric autosomes. Southern blot analyses revealed that this caribou satellite DNA sequence was well conserved in the genomes of five other deer species studied.In situ hybridization studies revealed Rt-Pst3-homologous DNA sequences in the centromeric regions of white-tailed deer chromosomes and Asian muntjac chromosomes, as well as at several interstitial chromosome regions in Indian muntjac chromosomes. Comparisons of the Rt-Pst3 DNA sequence to previously identified centromeric satellite DNA fragments from three other deer species revealed considerable DNA sequence similarity. The first ca. 800 bp of the Rt-Pst3 clone was found to share 73.8% similarity to theCCsatI clone of the European roe deer, 64.7% sequence similarity to the C5 DNA clone of the Chinese muntjac, and 64.8% and 65.6% sequence similarity to the 1A and B1 clones of the Indian muntjac, respectively. Moreover, the last 191 bp of the Rt-Pst3 clone was found to share about 60% DNA sequence similarity to the first 191 bp of the same clone. Amplification of one originalca. 800 bp monomer unit, along with the first 191 bp of the following juxtaposed monomer unit could have resulted in the tandemly repeated, 991 bp monomer unit now seen in the caribou genome. It is postulated that the centromeric satellite DNA found in other deer species, having repeat lengths greater than 800 bp, could also have evolved in a similar manner from a more ancestral monomeric unit ofca. 800 bp.     

Chromosomal localization of a single copy gene by in situ hybridization - human β globin genes on the short arm of chromosome 11

1. The localization of the β globin genes by in situ hybridization to fixed chromosomes is described.
2. The probe used was a [³H]cRN A copy of a genomic clone containing in total 4-4 kb of DNA and including the β globin gene.
3. The evidence for the localization of the gene comes from three pieces of data, ( a ) Chromosome 11 is labelled to double the extent expected if the grains were randomly distributed, ( b ) the extra grains above background are clustered on the short arm of 11 close to the centromere, and ( c ) the absolute number of grains observed is very close to that predicted for a probe of that length by comparison with ribosomal genes. The localization is in agreement with that obtained by other methods.
4. This method could be extended to any gene for which a genomic clone containing at least 5 kb of single copy DNA is available.     

Human insulin-related DNA sequences map to chromosomes 2 and 11

The insulin-related hormone family consists of four known peptides: insulin, the insulin-like growth factors I and II, and relaxin. To investigate the hypothesis that the family contains additional members, we have isolated a series of human genomic clones using the insulin gene as a hybridization probe. Two of these single copy DNA sequences, hIr 1 and hIr 2, have been localized to chromosome 2 and 11p11q13, respectively, by Southern blot analysis of mouse-human somatic cell hybrids, a result consistent with other evidence supporting the dispersal of the insulin gene family throughout the genome. Although a biological function for these DNA sequences has not yet been established, hIr1 and hIr2 are potentially useful as molecular probes for mapping, by analysis of restriction fragment length polymorphisms (RFLP), genetic disorders linked to chromosomes 2 or 11.     

Structure and chromosomal localization of the human 2',3'-cyclic nucleotide 3'-phosphodiesterase gene

Human brain cDNA clones for the myelin associated enzyme 2' 3' -cyclic nucleotide 3' -phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established.
The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.     

Localization of human U1 small nuclear RNA genes to band p36.3 of chromosome 1 by in situ hybridization

Ul small nuclear RNA (Ul snRNA) is encoded by a large family of genes (30–125 copies/haploid genome) which are transcribed by RNA polymerase II. Ul snRNA is thought to function in gene splicing. Since the Ul genes were found to be >20 kb apart by analyzing genomic phage clones, the chromosomal location of Ul genes in the human genome was determined using Southern filter analysis of DNA isolated from human-rodent somatic cell hybrids and by in situ hybridization. Human DNA digested with PvuII and probed with a Ul-specific probe, pD2, show several major hybridizing fragments. Of these, two human PvuII fragments of 1.4 kb and 2.4 kb had unique mobilities compared to mouse fragments. In a study of 19 cell hybrids, the human-specific Ul fragments segregated with the chromosome 1 markers peptidase C and adenylate kinase 2. All other chromosomes showed>-19% discordancy. An additional 13 karyotyped cell hybrids, analyzed by Southern filter analysis, confirmed the assignment of this class of Ul genes to chromosome 1. Additional digests with MspI and PstI indicated that most Ul genes are located on chromosome 1. To determine if the Ul RNAs are located predominantly at one site or dispersed over chromosome 1, a tritium-labeled Ul probe was hybridized in situ to metaphase chromosomes. The majority of the grains were at band 1p36.3, suggesting that most of the Ul genes are located in this region.A preliminary report of these experiments were presented at the Human Gene Mapping Workshop in Los Angeles, August 20–26, 1983 (1).