Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs

We have cloned a new species of rat Gi alpha (Gi3 alpha) cDNA and genomic DNAs for three distinct human Gi alpha proteins (Gi1 alpha, Gi2 alpha, and Gi3 alpha). Gi3 alpha cDNA codes for a protein of 354 amino acids (Mr 40,522) whose sequence is closely related but distinct from that of the previously isolated rat Gi alpha (Gi2 alpha). By screening the human genomic libraries with the two rat Gi alpha cDNAs as probes, clones encoding human Gi1 alpha, Gi2 alpha, and Gi3 alpha were isolated. The human Gi2 alpha and Gi3 alpha genes are composed of eight coding exons and seven introns and possess a completely identical exon-intron organization. Southern blot analysis indicates that a single copy of each Gi alpha gene is present per haploid human genome.     

Structure and expression of a newt cardio-skeletal myosin gene. Implications for the C value paradox

As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.     

Nucleotide sequence of cDNA clones coding for a brain-type fatty acid binding protein and its tissue-specific expression in adult zebrafish (Danio rerio)

We have determined the nucleotide sequence for two cDNA clones coding for a fatty acid binding protein (FABP) from zebrafish (Danio rerio). Comparison of the sequence with GenBank entries revealed extensive amino acid identity between this zebrafish FABP and brain FABPs (B-FABP) from other species. The zebrafish B-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases BglII or EcoRI suggesting that a single copy of the B-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish B-FABP mRNA is approximately 850 nucleotides in length. In situ hybridization revealed that the B-FABP mRNA was expressed in the periventricular gray zone of the optic tectum of the adult zebrafish brain.     

Isolation of major heat shock protein (hsp70) gene-related sequences from rat genome

Rat genome was assayed for the presence of hsp70 gene-related sequences. Southern blots prepared from rat DNA digested with EcoRI or HindIII restriction endonucleases were hybridized with mouse, human and fruit fly hsp 70 gene probes at increasing stringencies. At the stringency which allows sequences divergent up to about 30% to form stable complexes all three probes detected 25–30 restriction fragments. Increased stringency of the hybridization reduced the number of detectable bands to a few and among them the DNA fragments hybridizing specifically either with mouse or human hsp70 gene probes were detected. Most of the genomic fragments containing hsp70 gene-related sequences were subsequently isolated by screening the rat genomic library with mouse hsp70 gene probe. 168 positive clones were plaque purified and on the basis of the restriction and hybridization pattern we deduced that inserts represented 20 different genomic regions. Partial restriction maps of all isolated genomic fragments were constructed and regions containing hsp70 gene related as well as highly repetitive DNA sequences were localized. A putative sequence rearrangement in the proximity of the hsp70 gene-related sequence was detected in one of the isolated genomic segments.     

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.     

Isolation of a single nuclear gene encoding human ubiquinone-binding protein in complex III of mitochondrial respiratory chain

We have isolated five genomic DNA clones which contain nucleotide sequences hybridizable to a cDNA for human ubiquinone-binding protein in Complex III (QP). Nucleotide sequence analysis revealed that two of them contained different types of pseudogenes suggesting molecular evolution of the gene, and that the other three clones contained the overlapping fragments from the same QP gene. The gene spans 4.5 to 5 kb in length. The sequences of exons in the gene were determined and found to be identical to the corresponding parts of the human QP cDNA. The exon-intron boundaries follow the GT/AG rule. Two CAAT boxes were found in the promoter region. It is concluded from these results that the isolated human QP gene is functional. Genomic Southern blot analysis showed that the gene is present in a single copy in the human genome.     

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.     

Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis

A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined.     

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.     

Characterization of a new repetitive sequence in the Dictyostelium discoideum genome

A repetitive DNA sequence was isolated from a Dictyostelium discoideum genomic plasmid library of BglII-digested DNA ligated to the BamHI site in pBR322. This clone, called pBS582, hybridized to a large number of phage lambda Dictyostelium genomic clones. Southern blot analysis indicated that pBS582 DNA hybridized to many differently sized genomic DNA fragments generated by digestion with Eco RI, AvaI, or HindIII. Restriction maps of pBS582 and five genomic clones showed that the flanking regions of each of the genomic clones were different. These findings indicate that the sequence specific to pBS582 is scattered throughout the Dictyostelium genome and is reiterated approximately 100 times in the haploid genome. Northern blot analysis revealed that RNA which hybridized to pBS582 DNA was present during all stages of growth and development and did not seem to be developmentally regulated. Southern blot analysis of DNAs from other slime molds (D. giganteum, D. purpureum, and Polysphondylium violaceum) were performed to determine whether the pBS582 sequence was present in other species of slime molds. Hybridization of pBS582 was observed to DNA from the two Dictyostelium species but not to Polysphondylium. It may thus be possible to use hybridization of specific sequences as a biochemical tool to study the relatedness of different slime mold species and their molecular taxonomy.     

Characterization of the human cyclophilin gene and of related processed pseudogenes

The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.     

Structure of the gene for human butyrylcholinesterase. Evidence for a single copy

We have isolated five genomic clones for human butyrylcholinesterase (BChE), using cDNA probes encoding the catalytic subunit of the hydrophilic tetramer [McTiernan et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6682-6686]. The BChE gene is at least 73 kb long and contains four exons. Exon 1 contains untranslated sequences and two potential translation initiation sites at codons -69 and -47. Exon 2 (1525 bp) contains 83% of the coding sequence for the mature protein, including the N-terminal and the active-site serine, and a third possible translation initiation site (likely functional), at codon -28. Exon 3 is 167 nucleotides long. Exon 4 (604 bp) codes for the C-terminus of the protein and the 3' untranslated region where two polyadenylation signals were identified. Intron 1 is 6.5 kb long, and the minimal sizes of introns 2 and 3 are estimated to be 32 kb each. Southern blot analysis of total human genomic DNA is in complete agreement with the gene structure established by restriction endonuclease mapping of the genomic clones: this strongly suggests that the BChE gene is present in a single copy.     

Evolutionary aspects, structure, and expression of the rat interleukin 4 gene

The rat interleukin 4 (IL 4) gene has been isolated from a genomic lambda phage library by cross-hybridization to the mouse IL 4 cDNA. Like the mouse and human counterparts, it exists as a single copy gene in the genome and consists of four exons. The overall structure of the IL 4 locus seems highly conserved. This is indicated by the low degree of restriction fragment length polymorphism in a number of laboratory and wild mice and by the conservation of the intron size between human, rat, and mouse IL 4 genes. Furthermore, evolutionary conserved elements are the promoter region, the position of cysteine residues and sequence motifs in the 3' untranslated regions that are believed to be involved in destabilization of the mRNA. In contrast, the predicted amino acid sequence of the rat IL 4 gene shows low homology (57%) with the mouse homologue. The divergence between mouse and rat IL 4 genes is even more pronounced in the carboxy-terminal region (47% homology in the last 68 amino acids). The ratio between replacement and silent mutations in the IL 4 genes of different species suggests a complex pattern of selective forces acting on the IL 4 gene, which includes both selection against and for amino acid substitutions in individual positions. The functional identity with IL 4 has been confirmed by expression of the gene and the demonstration of the ability to induce MHC class II antigen expression on spleen cells.     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.