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.     

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.     

Nucleotide sequence of human rotavirus genome segment 10, an RNA encoding a glycosylated virus protein.

The complete nucleotide sequence of human rotavirus (Wa strain) genome segment 10 was determined by using a cloned DNA copy. The sequence data indicated that segment 10 is A + T rich (65%) and consists of 750 base pairs. The positive strand of segment 10 contains a single open reading frame that extends 175 codons from the first AUG triplet (residues 42 through 44). The amino acid sequence of the segment 10 product was deduced from the nucleotide sequence. There are two distinct glycosylation sites at the N-terminal hydrophobic region, consistent with previous findings that this protein exists in a glycosylated form. The apparent molecular weight (20,000) of the unglycosylated, precursor polypeptide is in good agreement with the one calculated from the predicted amino acid sequence. Structural analysis of the positive strand (mRNA from segment 10) showed that it could form, like mRNA from segment 11, a stable panhandle structure involving the 5' and 3'-terminal regions. The nucleotide sequence of segment 10 from simian rotavirus, recently determined by Both et al. (J. Virol. 48:335-339, 1983) was found to be highly homologous to, and to share several important features with, segment 10 of human rotavirus.     

5''-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency.

A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.     

Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain

The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleotide apex of the hairpin contains predominantly A and T residues and is incompletely base-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts at each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotide loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.     

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 minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting.

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.     

Analysis of the recombination event generating a vesicular stomatitis virus deletion defective interfering particle.

cDNA clones of different portions of the L cistron and 5'-terminal region of the vesicular stomatitis virus genome have been prepared and used to identify the exact site of the deletion in the defective interfering particle, DI-LT. The deletion extends from nucleotide 251 from the beginning of the L gene to a position 342 nucleotides from the end of the genome. The nucleotide sequences flanking the deletion site, as well as those at the ends of the deleted segment, did not contain any obvious vesicular stomatitis virus initiation or termination signals as had been found near the recombination sites in other defective interfering particle RNAs. The results best fit a model for the origin of this type of defective interfering particle in which the polymerase interrupts its synthesis and moves with its nascent daughter strand to a new position on the template and resumes synthesis there, further extending the nascent strand. Neither the interruption nor the resumption of synthesis appears to be in response to the template nucleotide sequence. The sequences of two partial L cistron clones also reveal open reading frames that code for amino acid sequences likely to be the amino and carboxy termini of the L protein.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Probable involvement of 3'-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A possibility of involvement of 3'-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 is accessible for complementary interactions in 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA when added to wheat germ cell-free protein synthesizing system were found to specifically inhibit translation of uncapped reporter mRNA coding for beta-glucuronidase, which bears in the 5'-untranslated region (UTR) a leader sequence of potato virus Y (PVY) genomic RNA possessing fragments complementary to the region 1777-1811. It was shown that a sequence corresponding to nucleotides 291-316 of PVY, which is complementary to a major portion of the 3-terminal 18S rRNA segment 1777-1808, when placed into 5'-UTR, is able to enhance translational efficiency of the reporter mRNAs. The results obtained suggest that complementary interactions between mRNA 5'-UTR and 18S rRNA 3'-terminal segment can take place in the course of cap-independent translation initiation.     

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.     

Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis.

The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.     

The suffix sequence is involved in processing the 3' ends of different mRNAs in Drosophila melanogaster

We describe here the nucleotide sequences of several genomic and mRNA copies of the suffix, a short dispersed actively transcribed repeat located at the 3' ends of many different genes of Drosophila melanogaster. Only one strand of the suffix is transcribed. The patterns of suffix-containing mRNAs vary during development. The five randomly selected genomic copies of the suffix are 265 bp long and quite conservative in their sequence. The non-transcribed strand is terminated with oligo(A) preceded by AATAAA sequence. No repetitive flanking sequences can be detected. The three other genomic copies selected by hybridization with suffix-containing cDNA clones are less conservative, especially in the 5' part. In particular, they contain short insertions carrying a polyadenylation signal AATAAA at exactly the same position of the suffix. Comparison of genomic and cDNA clones shows that mRNAs are polyadenylated at the last nucleotide of these insertions. The cDNA clones include the same part of the suffix, from the 39th to 112th nucleotide. Thus, a segment of the suffix forms the last exon for both genes. In one case, the beginning of the last intron coincides with the beginning of suffix, creating a very unusual donor splicing site. We conclude that the suffix sequence is directly involved in the formation of the last splicing site and 3'-end maturation of mRNA, at least in the case of the two genes analysed.     

Rat IGF-I cDNA's contain multiple 5'-untranslated regions

DNA sequencing of several independent rat IGF-I cDNA clones has revealed three different 5'-untranslated region sequences which contain multiple, upstream, in-frame initiation codons. Use of these codons could generate N-terminal heterogeneity in IGF-I precursor proteins. One of these 5'-untranslated region sequences contains a 40-bp segment which is an inverted repeat of a region in the common 3'-untranslated region. The ends of the IGF-I mRNA corresponding to this cDNA could form a stable duplex structure. Such a complex could prevent ribosomal access to the AUG codons preceding the coding region for the pre-pro-IGF-I peptide, suggesting the possibility of translational regulation of this form of IGF-I mRNA. The 3'-untranslated region inverted repeat sequence also is present in human and mouse IGF-I cDNA's, and, intriguingly, is more highly conserved than the rest of the 3'-untranslated region.     

Construction and characterization of cDNA clones encoding the 5' end of the chicken pro alpha 1(I) collagen mRNA

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.     

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.