Closely related families of genes code for the alpha and alpha' subunits of the soybean 7S storage protein complex

Nineteen cloned cDNAs encoding the alpha and alpha'-subunits of the 7S seed storage protein in the soybean, Glycine max, have been isolated from a recombinant cDNA library constructed with mRNA from maturing seeds. In addition, a gene encoding an alpha'-subunit has been isolated from a recombinant Charon 4A phage library containing genomic Glycine max DNA. The cloned DNAs have been divided, on the basis of their endonuclease sites, into two main classes of sequences which differ in approximately 6% of their nucleotides. Whereas the proteins encoded within each DNA class are nearly identical, the proteins encoded by the two different classes of soybean DNAs are distinct and correspond to alpha and alpha'-subunits. Thus, the alpha and alpha'-subunits are coded for by two closely related multigene families. The amino acid differences in the portions of the alpha and alpha'-subunits presented in this paper occur primarily near the carboxyl-terminus. The 3' noncoding nucleotides of the cloned alpha and alpha'-subunit DNAs are more highly conserved than are the coding nucleotides. This conservation suggests that the 3' untranslated sequences of the alpha and alpha'-subunit mRNAs are functional in the expression of the alpha and alpha'-subunit proteins or in the stabilization of the 7S subunit mRNAs.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.     

At least three alternatively spliced mRNAs encoding two alpha subunits of the Go GTP-binding protein can be expressed in a single tissue

Hybridization blot (Northern) analysis of mRNA coding for alpha subunits of the Go signal-transducing protein detects three bands at 5.7, 4.2, and 3.2 kilobases (kb). We showed previously that the largest is a splice variant coding for the type 2 form of the polypeptide (alpha o2) and the two smaller RNAs react with a probe specific for the seventh of the eight exons that code for the type 1 form (alpha o1). In the present work we demonstrate that the 3.2- and 4.2-kb mRNAs also result from alternative splicing, the splice site being located 31 nucleotides downstream from the termination codon of the open reading frame, and that therefore the alpha o mRNA is made up of at least nine exons. All three alpha o mRNAs are expressed in both heart and brain, more in the latter than the former, as well as in the hamster insulin-secreting tumor (HIT) cell from which the cDNAs encoding the splice variants had been cloned. In contrast, in lung and testis we found only the 5.7-kb alpha o2 mRNA. The same analysis was unable to detect alpha o-specific sequences in either kidney, pancreas (whole), spleen, or liver, while at the same time detecting strong bands for alpha s mRNA. A comparison of the nucleotide sequences of the 5'- and 3'-untranslated regions of the hamster cDNAs cloned here indicated that previously cloned alpha o cDNAs all belong to the same alpha o1A slice subclass derived from 3.2-kb mRNA. The comparison also revealed that the sequences of the untranslated regions are highly conserved among three species (rat, hamster, and brain). Their 3' tails are 99.1% (HIT versus bovine, 200 known bases) and 99.7% (HIT versus rat, 229 bases) identical, and their 5' leader sequences are 92.7% (HIT versus bovine, 165 known bases) and 90.7% (HIT versus rat, 670 bases) identical. This indicates that untranslated regions of mRNAs need not exhibit high degrees of species variation.     

alpha Subunit of rat pituitary glycoprotein hormones. Primary structure of the precursor determined from the nucleotide sequence of cloned cDNAs

Double-stranded complementary DNAs were constructed enzymatically from polyadenylated RNA extracted from pituitary glands of ovariectomized rats, were inserted into the Pst I site of plasmid pBR322 and were cloned in Escherichia coli chi 1776. Cloned cDNAs encoding the precursor to the alpha subunit (pre-alpha) of the glycoprotein hormones were identified by hybridization with a restriction fragment of a previously cloned and sequenced cDNA encoding the precursor to the alpha subunit of mouse thyrotropin (Chin, W. W., Kronenberg, H. M., Dee, P. C., Maloof, F., and Habener, J. F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5329-5333). The DNA sequences of the two largest rat cDNA inserts (591 and 554 base pairs) were determined and the amino acid sequence of the rat pre-alpha subunit was deduced from these sequences. The composite sequence determined from these cDNAs spans 610 base pairs, or almost the entire length of the messenger RNA (mRNA) of 800 bases, when account is taken of the 3' poly(A) tract. The rat alpha precursor consists of a 24 amino acid leader sequence and a 96 amino acid alpha subunit apoprotein. The amino acid homologies between the rat and mouse, and between the rat and human sequences are 95% and 74%, respectively. Nucleotide homologies between the rat and mouse cDNAs in the coding and untranslated regions are 94% and 80%, respectively. This cloned cDNA will be applied to analysis of the structure of the rat alpha subunit gene(s) and of the regulation of alpha subunit gene expression.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.     

Sequence of the yeast iso-1-cytochrome c mRNA

The nucleotide sequence of the yeast iso-1-cytochrome c (CYC1) mRNA is presented. The mRNA was enriched by hybridization to cloned CYC1 DNA attached to a solid matrix: either nitrocellulose filters or diazobenzyloxymethyl cellulose powder. The sequence of the 5'-end of the mRNA was determined by the extension of a CYC1-specific dodecanucleotide primer; the sequence of the 3'-end was determined using a decanucleotide d(pT8-G-A) primer. The CYC1 mRNA begins 61 nucleotides 5' to the AUG initiation codon, extends through the coding sequence to 172 to 175 nucleotides 3' to the UAA termination codon, followed by the poly(A) tail. There are no intervening sequences. Some of the sequences that the CYC1 mRNA shares in common with other eukaryotic mRNAs are discussed.     

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.     

Molecular basis for two forms of the G protein that stimulates adenylate cyclase

Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing.     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

Sequence determination and analysis of the 3' region of chicken pro-alpha 1(I) and pro-alpha 2(I) collagen messenger ribonucleic acids including the carboxy-terminal propeptide sequences

Three pro-alpha 1 collagen cDNA clones, pCg1, pCg26, and pCg54, and two pro-alpha 2 collagen cDNA clones, pCg 13 and pCg45, were subjected to extensive DNA sequence determination. The combined sequences specified the amino acid sequences for chicken pro-alpha 1 and pro-alpha 2 type I collagens starting at residue 814 in the collagen triple-helical region and continuing to the procollagen C-termini as determined by the first in-phase termination codon. Thus, the sequences of 272 pro-alpha 1 C-terminal, 260 pro-alpha 2 C-terminal, 201 pro-alpha 1 helical, and 201 pro-alpha 2 helical amino acids were established. In addition, the sequences of several hundred nucleotides corresponding to noncoding regions of both procollagen mRNAs were determined. In total, 1589 pro-alpha 1 base pairs and 1691 pro-alpha 2 base pairs were sequenced, corresponding to approximately one-third of the total length of each mRNA. Both procollagen mRNA sequences have a high G+C content. The pro-alpha 1 mRNA is 75% G+C in the helical coding region sequenced and 61% G&C in the C-terminal coding region while the pro-alpha 2 mRNA is 60% and 48% G+C, respectively, in these regions. The dinucleotide sequence pCG occurs at a higher frequence in both sequences than is normally found in vertebrate DNAs and is approximately 5 times more frequent in the pro-alpha 1 sequence than in the pro-alpha 2 sequence. Nucleotide homology in the helical coding regions is very limited given that these sequences code for the repeating Gly-X-Y tripeptide in a region where X and Y residues are 50% conserved. These differences are clearly reflected in the preferred codon usages of the two mRNAs.     

Restriction analysis of cDNA for cow alpha s1-, beta- and kappa-caseins

By means of in situ hybridization to cloned cDNA fragments coding for cow alpha s1-, beta- and kappa-caseins, screening of the library of clones containing the cDNA complementary to mRNA of lactating cow mammary gland was carried out. The clones containing the sequences of alpha s1-, beta- and kappa-casein cDNAs were shown to constitute 4.0, 3.2 and 0.7% of all the colonies, respectively. The analysis of the data on cross-hybridization points to the absence of extensive regions of homology between the above-mentioned cDNAs. The restriction analysis of cDNAs of the selected clones was carried out and the restriction maps of cDNAs of these three caseins were constructed. The restriction analysis data and determination of the nucleotide sequence of 5'-termini of the studied cDNAs indicated that the cloned sequences were the full-length mRNA copies of alpha s1-, beta- and kappa-caseins. The data obtained on restriction analysis are utilized in mapping the corresponding natural genes of cow caseins.     

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.     

The absence of the long 3'-non-translated region in mRNA coding for eye lens alpha A2-crystallin of the frog (Rana temporaria)

The nucleotide sequence of a cloned cDNA (clone pRt(1)297; GENE (1982) 17, 131) coding for a 18 kDa polypeptide of the frog eye lens has been determined. The sequence, 791 nucleotide in length has only one long open reading frame (447 nucleotides). The derived amino acid sequence in this frame has greater than 90% homology with the region 25-173 of alpha A2-crystallin amino acid sequence from a related frog species Rana pipiens. The 5'-terminal part of mRNA corresponding to the first 24 amino acids of alpha A2-crystallin has been lost in cloning and substituted by an artefactual sequence. The 3'-terminal part appears to be intact as follows from the presence of the universal poly(A) addition site and poly(A) tract. The 3'-nontranslated region present in frog alpha A2-crystallin mRNA (130 nucleotides) is about 4-times shorter than in mammalian alpha A2-crystallin mRNA. Intact alpha A2-crystallin mRNA with a size of about 700 nucleotides as determined by Northern blot hybridization is about twice smaller than corresponding mammalian mRNAs.     

Heterogeneity of polyadenylation site of mRNA coding for human serum albumin

Human fetal liver cDNA was cloned in pBR322 vector by dG:dC-tailing method. The cDNA library was screened for liver-specific clones by means of differential hybridization. Human fetal liver and human kidney cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand recombinants analysed. These clones represent cDNAs corresponding abundant mRNAs. Eighteen clones were identified as encoding serum albumin. Two different mRNA polyadenylation sites were found in four sequenced plasmids. Cleavage/polyadenylation site in two plasmids, pHA1 and pHA12, is situated fifteen nucleotides downstream the AATAAA signal; in two other plasmids, pHA8 and pHA25, this site is ten nucleotides downstream the same signal.     

Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA

A clone encoding the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) was isolated from a lambda gt11 expression library of rat brain cDNAs. The fusion protein expressed by the recombinant phage reacts with eIF-2 alpha antiserum except when the serum is preadsorbed with pure eIF-2. The translation of hybrid-selected HeLa cell mRNA produces two proteins which are indistinguishable from authentic HeLa eIF-2 alpha and its phosphorylated form when analyzed by electrophoresis in two-dimensional isoelectrofocusing/sodium dodecyl sulfate-polyacrylamide gels and by partial protease digestion. HeLa cell eIF-2 alpha mRNA migrates as a single band of about 1600 nucleotides. The rat cDNA insert was sequenced, and the region coding for eIF-2 alpha was identified. A human cDNA clone was obtained by hybridization screening with the rat cDNA, and its sequence was determined also. Both rat and human eIF-2 alpha proteins comprise 315 amino acids (36.1 kDa) and differ by only three amino acids. The eIF-2 alpha mRNA is found exclusively in polysomes containing 10 or more ribosomes in exponentially growing HeLa cells. In serum-depleted cells which synthesize eIF-2 and bulk protein more slowly than exponential cells, the level of eIF-2 alpha mRNA is not changed, the average polysome size is reduced to 7, and little or no eIF-2 alpha mRNA is detected in the ribonucleoprotein fraction. These results are consistent with the view that eIF-2 alpha mRNA translation is very efficient compared to other mRNAs in the cell.     

Cloning of a cDNA encoding an Artemia franciscana Na/K ATPase alpha-subunit.

Clones of cDNA that code for an isoform of the Artemia franciscana Na/K ATPase alpha subunit (NaKA alpha) have been isolated. The sequence of the longest of these clones (pArATNa136) is 3595 nucleotides; it codes for a 1004-amino acid protein whose sequence is identical to that of two previously sequenced Artemia NaKA alpha peptides. The encoded protein is over 73% identical to Drosophila melanogaster and vertebrate NaKA alpha s, and 73.8% identical to another Artemia NaKA alpha isoform previously described (named alpha 2850 in this article). The two Artemia cDNA clones code for mRNAs of different size; the clone pArATNa136 codes for a 4.5-kb mRNA while the alpha 2850 clone codes for a 3.6-kb mRNA. The degree of homology and the different size of the mRNAs encoded by both cDNAs suggest that they code for two different isoforms of the protein.