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Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.

Alpharetrovirus

Expression of murine dihydrofolate reductase complementary DNA in oocytes of Xenopus laevis.

1. Murine dihydrofolate reductase was produced efficiently in a Xenopus laevis oocyte that received microinjection of the dihydrofolate reductase mRNA. 2. Murine dihydrofolate reductase was produced under the direction of the plasmid pAdD26SVpA, which contains adenovirus major late promoter and the simian virus 40 T antigen gene for transcription initiation and murine dehydrofolate reductase complementary DNA fragment. 3. Injection of the plasmid pDHFR26, which contains murine dihydrofolate reductase complementary DNA and normal transcription initiation site segment in the 5' untranslational region, failed to produce proper translational products due to improper transcription.

Animals

Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II.

The calcium-calmodulin-dependent protein kinase II is a major component of brain synaptic junctions and has been proposed to play a variety of important roles in brain function. A complementary DNA representing a portion of the smaller 50-kilodalton subunit of the rat brain enzyme has been cloned and sequenced. The calmodulin-binding region has been identified and a synthetic analog prepared that binds calmodulin with high affinity in the presence of calcium. Like the 50-kilodalton kinase polypeptide, the concentration of the messenger RNA varies both neuroanatomically and during postnatal development of the brain. The broad tissue and species cross-reactivity of the complementary DNA suggests that the 50-kilodalton subunit found in rat brain is evolutionarily conserved and is the product of a single gene.

Age Factors

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.

Amino Acid Sequence

Molecular cloning and expression of a complementary DNA for inositol 1,4,5-trisphosphate 3-kinase.

A complementary DNA (cDNA) clone that encodes inositol 1,4,5-trisphosphate 3-kinase was isolated from a rat brain cDNA expression library with the use of monoclonal antibodies. This clone had an open reading frame that would direct the synthesis of a protein consisting of 449 amino acids and with a molecular mass of 49,853 daltons. The putative protein revealed a potential calmodulin-binding site and six regions with amino acid compositions (PEST regions) common to proteins that are susceptible to calpain. Expression of the cDNA in COS cells resulted in an approximately 150-fold increase in inositol 1,4,5-trisphosphate 3-kinase activity of these cells.

Amino Acid Sequence

The Cu(II)-repressible plastidic cytochrome c. Cloning and sequence of a complementary DNA for the pre-apoprotein.

We have cloned a complementary DNA for pre-apocytochrome c-552 from Chlamydomonas reinhardtii. The deduced sequence of the mature protein shows high homology to those of cytochromes c-553 from cyanobacteria. Its homology to mitochondrial cytochrome c or bacterial photosynthetic cytochrome c2 is lower and appears to be concentrated in sequences around amino acids involved in the interaction with heme. With respect to primary sequence, the "transit sequence" for cytochrome c-552 appears to show no homology to other transit sequences for nuclear encoded chloroplast proteins. However, based on analogy to transit sequences for other proteins (Daldal, F., Cheng, S., Applebaum, J., Davidson, E., and Prince, R. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2012-2016; Goldschmidt-Clermont, M., and Rahire, M. (1986) J. Mol. Biol. 191, 421-432; Smeekens, S., de Groot, M., van Binsbergen, J., and Weisbeek, P. (1986) Cell 46, 365-375) the transit sequence of cytochrome c-552 can be divided into envelope-traversing and thylakoid-traversing domains. Cytochrome c-552 appears to encoded by a single nuclear gene in C. reinhardtii. The gene is expressed exclusively in Cu(II)-deficient cells.

Amino Acid Sequence

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor.

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.

Amino Acid Sequence

Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.

Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

Avian Myeloblastosis Virus

Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide.

A cloned complementary DNA, termed pS2, was isolated from a human fibroblast cDNA library in the bacteriophage expression vector lambda gt11 after screening with a patient's serum containing a high titer of anti-ribonucleoprotein (RNP) antibodies. A reasonable amount of cro-beta-galactosidase fusion protein (pS2EX) was obtained through subcloning of the pS2 insert into a plasmid expression vector pEX-2. Antibody against pS2EX (anti-pS2EX) was purified from this patient's serum by Sepharose 4B conjugated with pS2EX. Immunofluorescent staining of HeLa cells with anti-pS2EX antibody exhibited a typical speckled pattern in the interphase nuclei. In the immunoblot analysis, the anti-pS2EX antibody recognized the 22 kDa protein. Using immunoprecipitation of cell lysate and subsequent RNA analysis, anti-pS2EX antibody was shown to precipitate U1 RNP only. The reactivities of various anti-RNP sera to pS2EX correlated well with the positive reaction to C polypeptide in the immunoblot. These findings indicate that pS2 is a cDNA for C polypeptide of U1 snRNP. In the Northern blot using human RNA and radiolabeled pS2, a single band about 800 base was observed. The nucleotide sequence of pS2 showed no significant homologies to known proteins.

Amino Acid Sequence

Molecular cloning and nucleotide sequence of complementary DNA encoding rabbit alpha 1-acid glycoprotein.

Alpha 1-acid glycoprotein (AGP) complementary DNA clone has been isolated from an acute phase rabbit liver library. Complete and full-length nucleotide sequence of this cDNA has been determined. The cDNA contains 35 nucleotides 5' untranslated region followed by 606 nucleotides of coding region and a 112 nucleotides 3' untranslated region. Comparison of this sequence with that of human, rat and mouse reveals a high degree of homology with human alpha 1-AGP. Northern blot analysis of messenger RNA for alpha 1-AGP in normal and acute livers demonstrates remarkable induction of transcription of this gene in rabbit in response to acute inflammation by the administration of turpentine. Such a response of AGP gene expression makes it a major member of the acute phase responsive genes in rabbit liver.

Amino Acid Sequence

Complementary DNA cloning and nucleotide sequence of rabbit serum amyloid A protein.

A complementary DNA clone encoding serum amyloid A protein has been isolated from an acute rabbit liver cDNA library. Complete nucleotide sequence analysis reveals that the cloned gene contains a 24 bases 5' untranslated region, 369 bases coding region and a 106 bases 3' untranslated region. Primer extension analysis indicates that the full-length 5' untranslated region contains 80 nucleotides. Northern blot analysis of mRNA from normal and acute rabbit livers demonstrates that this gene is expressed constitutively at a low level and undergoes induction of transcription in response to acute inflammation by the administration of turpentine.

Amino Acid Sequence

On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli.

The renaturation of complementary DNA strands by the recA protein of Escherichia coli has been found to exhibit the following features. (i) Optimal renaturation occurs at recA protein levels below that required to saturate the DNA strands; saturating amounts of recA protein significantly reduce the rate of reaction. (ii) The reaction proceeds in the absence of a nucleotide cofactor but is markedly stimulated by ATP in the presence of 10 mM Mg2+. A similar stimulation occurs in the absence of ATP when the Mg2+ concentration is increased from 10 mM to 30-40 mM. (iii) Both the ATP-stimulated and the Mg2+-stimulated reactions follow apparent first-order kinetics. These results, taken together with the known effects of ATP and Mg2+ on the state of aggregation of recA protein, suggest that the association of recA monomers may play an important role in recA protein-promoted DNA renaturation.

Adenosine Triphosphate

Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors.

Eleven complementary DNA (cDNA) clones were generated from messenger RNA isolated from abdominal light organs of the bioluminescent click beetle, Pyrophorus plagiophthalamus. When expressed in Escherichia coli, these clones can elicit bioluminescence that is readily visible. The clones code for luciferases of four types, distinguished by the colors of bioluminescence they catalyze: green (546 nanometers), yellow-green (560 nanometers), yellow (578 nanometers), and orange (593 nanometers). The amino acid sequences of the different luciferases are 95 to 99 percent identical with each other, but are only 48 percent identical with the sequence of firefly luciferase (Photinus pyralis). Because of the different colors, these clones may be useful in experiments in which multiple reporter genes are needed.

Adenosine Triphosphate

Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1).

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.

Amino Acid Sequence

Isolation and characterization of a complementary DNA (PD-1) differentially expressed by human pancreatic ductal cell tumors.

A complementary DNA (cDNA) library from a cloned subline (CD-11) of a well differentiated human pancreatic tumor cell line, HPAF, was subjected to differential screening using single stranded cDNA probes synthesized from mRNA of the well differentiated cell clone CD-11 and a poorly differentiated pancreatic tumor cell line, Panc 1. A cDNA clone (PD-1) was identified which had an insert of 626 base pairs (bp). PD-1 cDNA hybridized to a transcript of about 650 bp on Northern blot analysis, suggesting that the cDNA was close to full length. Densitometric analysis of Northern blots showed that a well differentiated pancreatic tumor line had a 5-fold higher PD-1 expression as compared to the poorly differentiated line, Panc 1. Nucleotide sequence analysis of the PD-1 cDNA and its deduced amino acid sequence showed an open reading frame of 399 bp. In addition to the open reading frame, the sequence had a 5' untranslated region of 61 bp and a 3' untranslated tail of 147 bp. The nucleotide sequence did not show any significant homology to any other sequence in the GENBANK or EMBL databases; however, the translated protein showed 35% homology to bacterial ribosomal proteins over 112 amino acids. Sequence analysis of the PD-1 cDNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its in vitro transcription/translation product suggest that this gene encoded a protein of 16,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA.

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.

Animals

A new growth-regulated complementary DNA with the sequence of a putative trans-activating factor.

A new complementary DNA (cDNA) clone has been isolated by differential screening of a cDNA library. The cognate RNA of this clone, called SC1, is growth regulated in human, mouse, and hamster cell lines. Its kinetics of growth regulation (time of increase in mRNA levels, sensitivity to cycloheximide, behavior in G1-specific temperature-sensitive mutants) classify the SC1 gene as a late growth-regulated gene, like the histone genes and the genes coding for the proteins of the DNA synthesis apparatus. By run-on assay, there is a modest increase in transcriptional rates after serum stimulation, which is not sufficient to explain the sharp increase in mRNA levels. The SC1 gene localizes to human chromosome 6p21-22. In bacteria, the SC1 cDNA clone makes a protein of Mr 39,000, in agreement with the putative reading frame. The amino acid sequence derived from the cDNA sequence indicates a previously unknown gene with a domain strongly suggestive of a trans-activating domain. The SC1 gene can be considered as coding for a possible new trans-activating factor that could play an important role in the transcription of genes required for the later stages of cell cycle progression.

Amino Acid Sequence

Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA.

Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.

Anemia, Sickle Cell