Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequences, the carboxyl-terminal peptide and the 3'-untranslated region are highly divergent and specific for this cDNA clone. There appears to be an amino acid deletion in the 3' end of the hamster alpha myosin heavy chain as compared to the rat alpha myosin heavy chain. S1 nuclease mapping experiments have shown that the mRNA represented by this cDNA clone is scarcely expressed in neonatal development, but its expression increases with age and reaches maximal levels in adult life. This cDNA clone provides a useful tool to follow the myosin heavy chain mRNA changes during development and during the genesis of a cardiomyopathy, an autosomal recessive defect carried by the Syrian hamster.     

Cloning and characterization of cDNAs encoding 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible rabbit mRNAs for cytochrome P-450 isozymes 4 and 6.

Monoclonal antibodies toward rabbit liver cytochrome P-450 isozyme 6 (P-450 6) were used to identify several recombinant clones from a pBR322 cDNA library that express beta-lactamase-P-450 6 hybrid proteins. The nucleic acid sequence and predicted amino acid sequence of a rabbit P-450 6 cDNA shows a high degree of homology with rat P-450c and mouse P1-450. When used as a probe to rescreen the library, the P-450 6 cDNA hybridized to several heterologous classes of cDNAs. One such class was shown to encode P-450 4 by comparison of its predicted amino acid sequence to amino acid sequences of cysteine-containing tryptic peptides derived from P-450 4. DNA sequence analysis of a cDNA clone belonging to a third class demonstrated that it contained a 131-base-pair intervening sequencing when compared to the cDNA coding for P-450 6. This sequence corresponds in location to intron E of the rat P-450c gene. Blot-hybridization analysis demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically induced P-450 4 and 6 mRNAs, which differ in size. The sizes of these mRNAs differ from their analogs in the mouse as a result of divergence in the 3' untranslated portions of the mRNAs.     

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.     

Extensive intragenic sequence homology in two distinct rat lens gamma-crystallin cDNAs suggests duplications of a primordial gene.

The nucleotide sequences of two different rat lens gamma-crystallin cDNA clones, pRL gamma 2 and pRL gamma 3, have been determined. pRL gamma 3 contains the complete coding information for a gamma-crystallin of 173 amino acids whereas pRL gamma 2 is incomplete in that it lacks the codons for the first three amino acids of a separate but very homologous gamma-crystallin of identical length. Both rat gamma-crystallins are homologous to the known amino acid sequence of bovine gamma-crystallin II which is only a single amino acid longer. The length of the region downstream the coding sequence to the A-A-T-A-A-A polyadenylylation signal sequence is 40 nucleotides in each clone. In pRL gamma 3 the poly(A) signal sequence is followed at 14 nucleotides by a remnant of the poly(A) tail which indicates that this clone contains a complete 3' noncoding region. pRL gamma 2 has only seven nucleotides following this signal sequence and no poly(A) tail, suggesting an incomplete 3' end. The cDNA clones show an overall nucleotide sequence homology of 85%. The mutual homology at the amino acid level is 73% whereas their amino acid homology with bovine gamma-crystallin II is about 70%. The nucleotide sequence of each clone also reveals a high intragenic homology and seems to be duplicated in itself. We suggest that the gamma-crystallin genes have arisen by multiple duplications of a primordial gene which consisted of about 120 nucleotides.     

Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit.

We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.     

Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat insulins I and II.

We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.     

Structure of two human alpha-tubulin genes.

The ability of a chicken alpha-tubulin cDNA probe to cross-hybridize with human DNA under stringent conditions has been exploited to screen two independently constructed human genomic libraries. Nine clones were isolated, accounting for 60% of the bands observed in a whole genomic Southern blot of human DNA. Two clones were selected for further analysis by restriction mapping, orientation experiments using 3'- or 5'-specific probes, and electron microscopy of heteroduplexes. One clone, 2 alpha, contains an alpha-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19 alpha, contains an alpha-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19 alpha has only two intervening sequences that correspond to the first two in clone 2 alpha. However, these intervening sequences differ in size between clones 2 alpha and 19 alpha and show no detectable sequence homology. The sum of the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length of the cDNA molecule.     

Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin.

We have synthesized two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin. Labeled oligonucleotides were used as hybridization probes to screen bacterial clones containing cDNA sequences primed with oligo(dT) and inserted into the plasmid vector pBR322. One beta 2-microglobulin cDNA clone was detected in the 535 bacterial plasmid clones that were screened. The clone has been characterized by blotting and nucleotide sequence analysis. The cloned beta 2-microglobulin sequence contains 217 base pairs of the 3' untranslated region of the mRNA and 328 base pairs (97%) of the coding region.     

Isolation and characterization of overlapping genomic clones covering the chicken alpha 2 (type I) collagen gene.

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.     

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.     

Sequence homology between RNAs encoding rat alpha-fetoprotein and rat serum albumin

We have determined the sequences of the recombinant DNA inserts of three bacterial plasmid cDNA clones containing most of the rat alpha a-fetoprotein mRNA. The resultant nucleotide sequence of alpha-fetoprotein was exhaustively compared to the nucleotide sequence of the mRNA encoding rat serum albumin. These two mRNAs have extensive homology (50%) throughout and the same intron locations. The amino acid sequence of rat alpha-fetoprotein has been deduced from the nucleotide sequence, and its comparison to rat serum albumin's amino acid sequence reveals a 34% homology. The regularly spaced positions of the cysteines found in serum albumin are conserved in rat alpha-fetoprotein, indicating that these two proteins may have a similar secondary folding structure. These homologies indicate that alpha-fetoprotein and serum albumin were derived by duplication of a common ancestral gene and constitute a gene family.     

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5''-specific genomic clones.

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.     

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.     

Human cardiac myosin heavy chain genes. Isolation of a genomic DNA clone and its characterization and of a second unique clone also present in the human genome

A gene sequence coding for myosin heavy chain (MHC) of human cardiac muscle was isolated by screening a human genomic library with a 32P-labelled 1.1kb SacI restriction fragment from a previously characterized cDNA clone specifying the light meromyosin and 3' untranslated region of mRNA encoding rabbit cardiac alpha-MHC. The DNA of this human genomic clone (lambda HCMHC8) hybridized much more strongly than did other clones isolated under similar, low stringency conditions both to the rabbit cDNA probe and to mRNA isolated from rat cardiac, but not skeletal, muscle tissue. Probe made from a DNA restriction fragment of lambda HCMHC8 hybridized a single 31S band of human ventricular mRNA. This size is identical to that of cardiac MHC mRNA of other species. Heteroduplex analysis showed hybridization of lambda HCMHC8 with exon segments in a rabbit cardiac MHC genomic clone (lambda MHC alpha 12/1). It also showed that lambda HCMHC8 spanned 14 kb of DNA and contained exon segments estimated to code for two-thirds of a MHC including the carboxylic acid terminus. By rescreening the library under more stringent conditions, where only DNA sequences having strong homology to cardiac MHC genes would be expected to hybridize, clones having restriction maps overlapping lambda HCMHC8 were isolated together with a unique clone (lambda HCMHC9). DNA gel blot hybridization of human genomic DNA with lambda HCMHC8 probe at medium stringency gave a pattern of restriction fragments similar to the restriction map of lambda HCMHC8. A weaker set of bands also appeared which corresponded in pattern to the map of lambda HCMHC9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain.

A cDNA library derived from human cerebral cortex was screened for the presence of sodium channel alpha subunit-specific clones. Ligation of three overlapping clones generated a full-length cDNA clone, HBA, that provided the complete nucleotide sequence coding for a protein of 2005 amino acids. The predicted structure suggests four homologous repeats and exhibits greatest homology and structural similarity to the rat brain sodium channel II. A second cDNA clone, HBB, that encodes a different subtype of sodium channel was isolated. Hybridization of DNA fragments from the 3' untranslated region of HBA and PCR with primers derived from HBB with human-hamster somatic cell hybrids localized these clones to human chromosome 2. In situ hybridization to human metaphase chromosomes mapped the structural genes for both HBA and HBB sodium channels to chromosome 2q23-24.3. The sodium channel HBA gene product was expressed by transfection in CHO cells. Expressed HBA currents were voltage-dependent, sodium-selective, and tetrodotoxin-sensitive and, thus, exhibit the biophysical and pharmacological properties characteristic of sodium channels.     

Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.

Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.