Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

A common myosin light chain is expressed in chicken embryonic skeletal, cardiac, and smooth muscles and in brain continuously from embryo to adult

We have isolated cDNA clones of the mRNA for chick embryonic myosin light chain (MLC), L23, by cross-hybridization with chicken skeletal muscle MLC1 cDNA. The identification of the isolated cDNAs was carried out by in vitro translation of hybrid-selected mRNA. Sequence analysis of the cloned cDNAs revealed that the cDNA insert contained 832 nucleotides and predicted a polypeptide of 185 amino acids with a calculated molecular weight of 20,687. The deduced amino acid sequence for L23 showed high sequence similarities to those of adult alkali type MLCs from various tissues, indicating that L23 belongs to the alkali MLC group. Using the cloned cDNA as a hybridization probe, we have revealed by RNA blot analysis that the expression of L23 mRNA was regulated in temporal and tissue-specific manners. The L23 mRNA of 1.1 kilobases is transiently expressed in embryonic skeletal, cardiac, and smooth muscles of chickens. It is also found in the brain of chickens during all stages of development so far investigated. Only a single gene for L23 was detected by Southern blot of chick genomic DNA. We therefore suggest that L23 is expressed from a single gene in both embryonic muscles and brain.     

Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II

A cDNA clone for human carbonic anhydrase (CA) II was isolated from a kidney lambda gt10 library. Expression of the cDNA insert in Cos-7 cells produced an immunoprecipitable product and enzymatically active carbonic anhydrase. The cDNA insert is 1551 bp in length and contains an open reading frame which encodes a 260-amino-acid polypeptide. The deduced amino acid sequence is identical to that reported for human CA II. The protein coding region of this cDNA for human CA II shows 81 and 70% nucleotide identity with cDNAs for CA II from mouse and chick, respectively. Even the long 3'-untranslated region of the cDNA for human CA II (703 bp) is 64 and 42% identical to those of CA II from mouse and chick, showing remarkable conservation of the CA II cDNAs in amniotes. The protein coding region of the human CA II cDNA is 64 and 65% identical with those of human CA I and CA III, which are thought to have arisen from a common precursor by gene duplication.     

Isolation and characterization of variant cDNAs encoding mouse tyrosinase

Two different cDNA clones encoding mouse tyrosinase (monophenol oxygenase, E.C. 1.14.18.1) were isolated from B16 melanoma cells, and their primary structure was determined. One of the cDNAs consists of 3309 nucleotides with an open reading frame coding for a peptide of 533 amino acids. The other cDNA is approximately 1600 nucleotides long, with a shorter 3'-untranslated region and a deduced in-frame deletion of 77 amino acid residues with respect to the former clone. Neither of these clones is structurally identical to other described mouse tyrosinase cDNAs (1-3). RNA blotting analysis demonstrates that multiple tyrosinase mRNA species are not only present in B16 melanoma, but also in normal skin melanocytes.     

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.     

Molecular cloning and expression of a novel adhesion molecule, SC1

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.     

Molecular cloning of retinal oxidase/aldehyde oxidase cDNAs from rabbit and mouse livers and functional expression of recombinant mouse retinal oxidase cDNA in Escherichia coli

Retinal oxidase (EC 1.2.3.11) is a molybdenum-containing flavoenzyme with high enzymatic activity as to retinoic acid synthesis. In this study, we provide direct evidence that retinal oxidase is identical to aldehyde oxidase (EC 1.2.3.1) by cDNA cloning. Retinal oxidase and aldehyde oxidase, purified from rabbit liver cytosol using the original methods, showed completely identical HPLC patterns and amino acid sequences for three corresponding polypeptides (103 amino residues). The primary structural information obtained from the cleaved polypeptides permitted molecular cloning of the full-length cDNA of rabbit liver retinal oxidase (aldehyde oxidase). We also cloned and sequenced the full-length cDNA of mouse retinal oxidase. The cDNAs of rabbit and mouse retinal oxidase have a common sequence approximately 4.6 kb long, comprising 4-kb coding regions. The open reading frames of the cDNAs predict single polypeptides of 1334 and 1333 amino acids; the calculated minimum molecular mass of each is approximately 147,000. Northern blot analysis showed that the rabbit retinal oxidase mRNA was widely expressed in tissues. Finally, we successfully constructed a prokaryotic expression system for mouse retinal oxidase. The purified recombinant retinal oxidase from Escherichia coli showed a typical spectrum of aldehyde oxidases and a lower Km (3.8 microM) for retinal and a higher Vmax (807 nmol/min/mg protein) for retinoic acid synthesis than those of rabbit retinal oxidase (8 microM and 496 nmol/min/mg protein). This represents the first eukaryotic molybdenum-containing flavoprotein to be expressed in an active form in a prokaryotic system.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Alternative splicing of glucokinase mRNA in rat liver.

The sequences of two near full-length cDNAs encoding rat liver glucokinase are reported. One of the cDNAs is essentially identical to the cDNA cloned by Andreone, Printz, Pilkis, Magnuson & Granner. [(1989) J. Biol. Chem. 264, 363-369]. The other cDNA contains a 151 bp insertion and a downstream 52 bp deletion. The inserted block of bases has been shown to originate from an optional cassette exon, termed 2A, between the previously described exons 1 and 2. The conceptual translation product from the variant mRNA is identical to the original glucokinase protein for the first 15 amino acids. Next there is a novel polypeptide sequence of 87 residues, comprising 50 residues encoded by the cassette exon and 37 residues specified by an altered reading frame in exon 2. Due to the 52 bp deletion, 17 amino acids of the reference sequence are then missing, after which the sequence reverts to the original. Northern blot analysis with oligonucleotide probes has shown that alternatively spliced mRNA represents about 5% of total glucokinase mRNA. Alternative splicing of glucokinase mRNA in liver may explain earlier findings of minor isoforms of hepatic glucokinase.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Sequence of carbonic anhydrase II cDNA from chick retina

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes.

We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.     

Characterization of a cloned cDNA encoding rabbit myelin P2 protein

Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.