Molecular cloning and sequencing of a murine pgk-1 pseudogene family

Seven genomic mouse DNA fragments carrying pgk-1-homologous regions have been cloned and sequenced. They have to be classified as processed genes because intervening sequences, present in their productive counterpart, are absent. Four pseudogenes (I-IV) represent nearly the complete sequence of pgk-1 cDNA. Two of these genes (I and II), although rather different from the published mouse pgk-1 cDNA in the 3'-untranslated region, represent the actual mouse pgk-1 cDNA sequence in the coding part except for substitutions in the third position of three codons. These genes can code for a functional PGK protein but, lacking as they do classical promoter structures, are probably not expressed. They show the typical characteristics of retroposons, being flanked by A-rich regions and direct repeats which are localized at the positions where the homology with the mouse pgk-1 cDNA is interrupted. Pseudogenes III and IV have numerous mutations. Gene III is also flanked by direct repeats, whereas gene IV is flanked by inverted repeats. The other three genes are flanked by direct repeats localized further inside the target sites. They are truncated and mutated extensively as usually observed with pseudogenes.     

Molecular cloning, sequencing, and chromosome mapping of a 1A-encoded omega-type prolamin sequence from wheat.

Gliadins are the most abundant component of the seed storage proteins in cereals and, in combination with glutenins, are important for the bread-making quality of wheat. They are divided into four subfamilies, the alpha-, beta-, gamma-, and omega-gliadins, depending on their electrophoresis pattern, chromosomal location, and DNA and protein structures. Using a PCR-based strategy we isolated and sequenced an omega-gliadin sequence. We also determined the chromosomal subarm location of this sequence using wheat aneuploids and deletion lines. The gene is 1858 bp long and contains a coding sequence 1248 bp in length. Like all other gliadin gene families characterized in cereals, the omega-gliadin gene described here had characteristic features including two repeated sequences 300 bp upstream of the start codon. At the DNA level, the gene had a high degree of similarity to the omega-secalin and C-hordein genes of rye and barley, but exhibited much less homology to the alpha- and beta-gliadin gene families. In terms of the deduced amino acid sequence, this gene has about 80 and 70% similarity to the omega-secalin and C-hordein genes, respectively, and possesses all the features reported for other gliadin gene families. The omega-gliadin gene has about 30 repeats of the core consensus sequences PQQPX and XQQPQQX, twice as many as other gliadin gene families. Southern blotting and PCR analysis with aneuploid and deletion lines for the short arm of chromosome 1A showed that the omega-gliadin was located on the distal 25% of the short arm of chromosome 1A. By comparison of PCR and A-PAGE profiles for deletion stocks, its genomic location must be at a different locus from gli-Ala in 'Chinese Spring'.     

Xp22.3; Yq11.2 chromosome translocation and its clinical manifestations

We report clinical and molecular investigations in a boy with karyotype 46,Y,der(X)t(X;Y)(qter-->p22.3::q11.21-->qter) and his mother with karyotype 46,X,der(X)t(X;Y)(qter-->p22.3::q11.21-->qter). Haplo-insufficiency for the Xp22.3-->pter chromosomal region in the boy resulted in postnatal growth retardation, developmental delay, partial ichthyosis and facial dysmorphism, but normal external genitals. His mother has a normal phenotype with normal stature and gonadal function but borderline intelligence. FISH-analysis showed a duplication of the Y-heterochromatin probe in the proband and a deletion of the Y933D4 probe in his mother. Molecular investigations situated the Xp22.3 breakpoint between DXS278 and the KAL gene and the Yq11.21 breakpoint between the DYS391 and DYS390 in the proband and his mother. X-inactivation study was performed by analysis of the polymorphic CAG-repeat in the androgen-receptor gene as described showing a normal random (40% versus 60%) inactivation pattern in the mother. The manifestations in male and female with loss of the Xp22.3-->pter and gain of the Yq11.21-->qter chromosomal region are discussed.     

A third neurofibromatosis type 1 (NF1) pseudogene at chromosome 15q11.2

Sequences related to the neurofibromatosis type 1 (NF1) gene have been identified on several human chromosomes. In the centromeric region of chromosomes 14 and 15, two NF1 pseudogenes have been described. Sequence comparison between NF1-related exons amplified from two yeast artificial chromosome clones hybridizing to chromosomal region 15q11.2 and published NF1-related sequences localized at 15q11.2 suggested that a third NF1 pseudogene resides in this chromosomal region. The previous localization of an NF1-related locus to the telomeric part of chromosome 15 could not be confirmed by us. Our findings further support pericentromeric spreading of partial NF1 gene copies at chromosome 15q11.2 during evolution. Received: 27 January 1996 / Accepted: 26 May 1997     

Molecular cloning of mouse beta 2-glycoprotein I and mapping of the gene to chromosome 11.

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.     

Molecular cloning and mapping of a novel ADAM gene (ADAM29) to human chromosome 4

Members of the ADAM family (type I integral membrane protein with a disintegrin and metalloprotease domain) have been implicated in many important biological processes involving cell-cell and cell-matrix interactions, such as fertilization and myoblast fusion. We report here the cDNA sequence of a novel human ADAM gene (ADAM29) that contains a putative fusion peptide. Northern blot analysis revealed that the mRNA of ADAM29 is highly expressed in the testis. By radiation hybrid panel mapping, the ADAM29 gene was assigned to human chromosome 4q34.2-qter.     

Molecular cloning, chromosomal localization, and expression of the murine SALL1 ortholog Sall1

SALL1 has been identified as one of now three human homologs of the region specific homeotic gene spalt (sal) of Drosophila, which encodes a zinc finger protein of characteristic structure. Mutations of SALL1 on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS, OMIM no. 107480). In order to facilitate functional studies of this gene in a model organism, we searched for the murine homolog of SALL1. Here we report the genomic cloning, chromosome mapping, and partial expression analysis of the gene Sall1. Sequence comparison, Northern blot hybridization as well as the conserved chromosome location on the homologous mouse chromosome indicate that we have indeed isolated the murine homolog of SALL1.     

Assignment of human prochymosin pseudogene to chromosome 1

Chymosin is an extremely specific aspartatic protease responsible for milk coagulation. Chymosin is expressed in a number of mammalian offspring, yet its presence in the gastric tissue of human infants remains a matter of controversy. In any event, the human genome contains chymosin-related sequences that probably represent a pseudogene. Using DNA obtained from human x hamster somatic cell hybrids as the template and polymerase chain reaction, we have mapped the human prochymosin pseudogene to chromosome 1.     

Molecular cloning, chromosome mapping and characterization of a testis-specific cystatin-like cDNA, cystatin T

The cystatin superfamily of cysteine proteinase inhibitors consists of three major families. In the present study, we report the cloning of the cDNA for mouse cystatin T, which is related to family 2 cystatins. The deduced amino acid sequence of cystatin T contains regions of significant sequence homology including the four highly conserved cysteine residues in exact alignment with all cystatin family 2 members. However, cystatin T lacks some of the conserved motifs believed to be important for inhibition of cysteine proteinase activity. These characteristics are seen in two other recently cloned genes, CRES and Testatin. Thus, cystatin T appears to be the third member of the CRES/Testatin subgroup of family 2 cystatins. The mouse cystatin T gene was mapped on a region of chromosome 2 that contains a cluster of cystatin genes, including cystatin C and CRES. Northern blot analysis demonstrated that expression of mouse cystatin T is highly restricted to the mouse testis. Thus, a shared characteristic of the cystatin family 2 subgroup members is an expression pattern limited primarily to the male reproductive tract.     

Molecular cloning and characterization of a high affinity dopamine receptor (D1 beta) and its pseudogene.

We have cloned a novel human intronless gene encoding a G-protein-coupled receptor of the dopamine receptor family. Expression of this receptor in Cos-7 cells led to the high affinity binding of a number of dopamine D1 antagonists, with a binding profile similar to that of the previously described dopamine D1 receptor. In contrast, the agonist binding profile of this new receptor did not exactly match any previously defined dopamine D1 receptor and was notable for its unusually high affinity for dopamine. This new receptor caused a 13-fold increase in adenylylcyclase activity in transfected Cos-7 cells, following addition of dopamine. Messenger RNA encoding this new receptor appears to be widely distributed in the human brain, including cortical regions, choroid plexus, hippocampus, and brain stem. This new receptor appears to be identical to the recently described dopamine D5 receptor. A second closely related gene, GL39, was isolated and shown to represent a pseudogene, the first to be described in the G-protein-coupled receptor superfamily. This pseudogene exhibits 94% nucleotide sequence homology to the GL30 sequence and may have arisen from a gene duplication event followed by a mutation approximately 8 million years ago, prior to the emergence of man. This recently evolved pseudogene is transcribed in the human brain with a tissue distribution similar to that for its closely related functional gene.     

Fine mapping of a human chromosome 6 ferritin heavy chain pseudogene: relevance to haemochromatosis

Summary We have used a somatic cell hybrid regional mapping panel for the short arm of chromosome 6, linkage analysis and a population study to map in detail a previously described ferritin heavy chain pseudogene sequence on chromosome 6. Our results show that this sequence maps to the short arm of chromosome 6 centromeric to the glyoxylase locus. The ferritin pseudogene locus is thus distant from the locus for the iron storage disease haemochromatosis, confirming previous evidence that this sequence is not a candidate for the haemochromatosis gene.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain

The sequence of the ubiquitin protein is highly conserved between species and has facilitated the cloning of numerous ubiquitin-like proteins. In the present study, we report the cloning of the cDNA for human ubiquilin 3 (UBQLN3). The deduced amino acid sequence of UBQLN3 contains a UBQ domain (ubiquitin-like) in the amino terminus as well as two highly conserved domains found in several recently cloned ubiquitin-like proteins. One of these domains, termed the NP domain, is a highly conserved 93 amino acid region present in UBQLN3 and several ubiquitin-like proteins. The last conserved domain is the UBA domain (ubiquitin-associated) found in a variety of proteins of the ubiquination pathway. The human UBQLN3 gene was mapped to the 11p15 region of chromosome 11. Northern blot analysis of multiple human and mouse tissues demonstrated UBQLN3 mRNA expression specifically in testis.     

Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line.

Molecular analysis of Saccharomyces cerevisiae secretion mutants has led to the identification of two Ras-like GTP-binding proteins, Ypt1p and Sec4p, which are essential for transport along the exocytic route. To study the regulation of membrane traffic in epithelial cells, a set of 11 clones encoding proteins similar to the YPT1/SEC4 products were isolated from an MDCK (Madin-Darby canine kidney) cell cDNA library. Four of these proteins, Rab8, -9, -10, and -11, are novel members of this subfamily of Ras-like proteins, and two of them are closely related to Ypt1p and Sec4p. The ratio of the number of clones isolated over the total number screened reveals a high level of complexity for this subfamily of GTP-binding proteins. This diversity supports their proposed function in controlling different steps in membrane traffic.