Complementary DNA sequence of a human cytoplasmic actin. Interspecies divergence of 3' non-coding regions

We have isolated and sequenced a cloned complementary DNA insert complementary to the messenger RNA of a cytoplasmic actin expressed in human epidermal cells. This provides the first cytoplasmic actin complementary DNA sequence for a vertebrate organism. The actin amino acid sequence predicted from this complementary DNA is identical to that of a bovine cytoplasmic actin and shows 98 and 85% homology with a Dictyostelium and a yeast actin, respectively. The complementary DNA sequence indicates that the 3' end of the mRNA contains an unusually long (greater than 400 nucleotides) 3' non-translated region. A comparison of this 3' non-coding region with those of recently determined actin complementary DNA sequences from other species reveals little or no homology among these sequences. Thus, these results indicate that although the actin amino acid sequences are extremely conserved, the non-coding regions of the mRNAs diverge rapidly.     

The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full-length clone includes 21 bp at the transcribed non-coding 5' region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non-coding 3' region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane-spanning segments. Using plasmid-programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.     

An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.     

Structure of the black beetle virus genome and its functional implications

The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5' non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3' proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.     

Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constatn region of kappa chain in the mouse

The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the cDNA² contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large cDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3′ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5′ terminus, this cDNA would code for 93% of the entire kappa chain.The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli.The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity.     

Cloning and sequence analysis of a cDNA for the alpha-globin mRNA of carp, Cyprinus carpio

A cDNA for alpha-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161-170) and its complete nucleotide sequence was determined. The 5' non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an alpha-globin polypeptide consisting of 142 amino acids. The 3' non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp alpha-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.     

Molecular cloning of yeast cytochrome C-like polypeptide expressed in human lung carcinoma: An antigen recogizable by lung cancer-specific human monoclonal antibody

Summary We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c fromCandida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. Thein vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297–475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.     

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.     

Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases

Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.     

The sequence at the 3'' terminus of mouse immunoglobulin secreted mu chain messenger RNA determined from cloned cDNA

The 3' terminal nucleotide sequence of two clones containing DNA complementary to mu chain mRNA of IgM-secreting cells has been determined. The sequence shows a termination codon (UGA) adjacent to the terminal tyrosine codon for the secreted protein and a 3' non-coding region of at least 106 bases. The primary translation product of this mu chain mRNA seems to terminate at the tyrosine of the secreted protein.     

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310. The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.