Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

The analysis of a chicken myosin heavy chain cDNA clone

A cDNA library has been constructed in the plasmid pBR322 using a large size class of RNA derived from chicken embryonic leg muscle as the template material. A clone containing a 2350-base pair insert was selected and identified as coding for the myosin heavy chain sequence, based upon its ability to hybridize to genomic myosin heavy chain clones, and by direct nucleotide sequencing. Cross-hybridization experiments with myosin heavy chain genomic clones, and mRNAs derived from different muscle types were used to explore the heterogeneity of the various myosin heavy chain isoforms at the level of the coding sequences. Although extensive sequence homology with the other isoforms was observed, a fast white isoform-specific subclone was constructed, and used to demonstrate that different genes code for the adult and embryonic fast white myosin heavy chain proteins.     

Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5'' exons and possible mechanism for the genesis of the 3'' end of v-src.

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.     

Wheat Ec metallothionein genes. Like mammalian Zn2+ metallothionein genes, wheat Zn2+ metallothionein genes are conspicuously expressed during embryogenesis.

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P-labeled oligonucleotide probes that encoded parts of the partial amino-acid sequence for the Zn-containing Ec protein. Each DNA insert in 11 positives from a screen of 10(5) plaques encoded a 5' untranslated and a 3' untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the Ec protein. The three different mRNA sequences encoded in the cDNA probably correspond to single-copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P-labeled cDNA and gave a single positive in a screen of 5 x 10(5) plaques. A 3.1-kb genomic fragment (gf-3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf-3.1 sequence encodes an intronless mRNA for the Ec protein and contains appreciable amounts of 5' and 3' flanking sequences. In addition to a putative TATA box, two inverted-repeat sequences and one direct-repeat sequence, the 5' flank in gf-3.1 contains a sequence similar to the abscisic-acid-responsive element in other higher-plant genes but does not contain sequences similar to the metal-responsive elements in animal metallothionein genes. Consistent with these findings, RNA blotting shows that accumulation of Ec mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat Ec metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.     

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

Cloning of the cDNA encoding a neuronal myosin heavy chain from mammalian brain and its differential expression within the central nervous system.

The complete amino acid sequence of a neuronal myosin heavy chain (MHC) from mammalian brain (1999 amino acids, 230 kDa) has been deduced by sequencing cDNA clones isolated from a rat brain cDNA library. The library was screened using an affinity-purified polyclonal antibody that had been raised against myosin purified from a neuronally-derived cell line (Neuro-2A). Restriction digests of genomic DNA from Neuro-2A cells and rat brain are consistent with an identity of the sequenced isoform from these two sources. RNA blot analysis demonstrates this myosin to exhibit differential expression within the cerebral cortex and spinal cord. No expression was observed in liver, kidney, heart, spleen or skeletal muscle, or even within other regions of the brain. The sequence of this neuronal MHC is compared with those of other non-muscle MHCs, to which it shows an overall similarity of structure, especially with respect to conserved regions within the head (ATP binding site, actin binding site, reactive thiols) and the presence of an alpha-helical coiled-coil tail that can be arranged as 28-residue repeating units plus four skip residues. A unique non-helical tailpiece composed of 72 amino acid residues marks the C-terminus of this neuronal myosin isoform.     

Myosin heavy chain accumulates in dissimilar cell types of the macromere lineage in the sea urchin embryo

In this study, myosin heavy chain from sea urchin pluteus larvae was characterized by analysis of a 2.5-kb cDNA clone. DNA sequence of 1465 bp demonstrated a 71% similarity in the deduced amino acid sequence to the embryonic rat skeletal muscle sequence. Antibodies generated against a polypeptide encoded by the open reading frame of the cDNA clone specifically identified a 210-kDa myosin protein which accumulated in 8-12 muscle cells differentiating bilateral to the esophagus beginning at early larval stages. This same myosin also accumulated in cells of the endodermal epithelium that comprise the three sphincters of the larval gut. Thus, a gene encoding myosin heavy chain is expressed in dissimilar cell types of the macromere lineage, and the pattern of accumulation in the gut identifies functionally distinct cells of the endodermal epithelium.     

Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Two genes encode the two subunits of cottonseed catalase

The isolation and sequence of a cDNA encoding a developmentally distinct subunit of cottonseed catalase are presented. A 1.8-kb cDNA was selected from a cDNA library constructed with poly(A)+ RNA isolated from 3-day-old dark-grown cotyledons in which a second subunit (designated SU 2 in an earlier publication) of catalase was predominantly synthesized. The cDNA encodes a 492-amino acid peptide with a calculated Mr of 56,900. The nucleotide sequence is 76% identical to a cDNA encoding another subunit (SU 1) which was predominantly synthesized in 1-day-old-cotyledons. Most of the divergence occurs in the 5' and 3' non-coding regions, and at the third positions of the codons. The deduced amino acid sequence is 92% identical to that of SU 1. Denaturing isoelectric focusing and SDS-PAGE of products transcribed and translated in vitro from these cDNAs revealed that the cDNA selected from the "1-day" library encoded SU 1 and the cDNA selected from the "3-day" library (this paper) encoded SU 2 of catalase. These data and results from Southern blot analyses of genomic DNA indicate that there are two genes encoding catalase subunits in cotton cotyledons, with only one copy of SU 1 and at least two copies of SU 2 in the genome. A peroxisomal targeting signal, e.g., Ser-Lys-Leu, is not located at the C-terminus of either subunit, or within 25 residues of the C-terminus of SU 1, although it occurs at six residues upstream from the C-terminus of SU 2. A possible location of a targeting sequence for catalase and other peroxisomal proteins lacking the C-terminal tripeptide motif is proposed.     

Cloning and sequence determination of a complementary DNA related to human liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.