Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae.

The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of vacuolar H(+)-ATPases: organellar and tissue distribution of 16 kDa proteolipids.

A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15,807 Da (pI = 9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H(+)-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Characterization of a cDNA clone for a rare mRNA modulated by ovariectomy in mammary carcinomas

A complementary DNA (cDNA) clone (p13) for a rare mRNA was isolated from a cDNA library generated from total polyA+ RNA of 14-day lactating rat mammary gland. In vitro translation of the positively selected mRNA from p13 cDNA revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide of 24 kDa. The p13 cDNA clone hybridized on northern blots predominantly to approximately 1100 base size RNA and weakly to approximately 3800 base size RNA from lactating mammary gland. It hybridized only to approximately 3800 base size RNA from rat liver. Southern blot analysis of genomic DNA showed differences in gene organization in mammary gland and liver. The mRNA level for the 24 kDa polypeptide was higher in 7-12 DMBA-induced tumor and lower in the MTW9 carcinoma as compared to lactating mammary gland. After ovariectomy, the mRNA level in mid pregnant gland increased but was reduced in the 7-12 DMBA tumors.     

Molecular cloning of the cDNA encoding an immunodominant antigen of Dirofilaria immitis.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 kDa antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a lambda gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consisted of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34,400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.     

Human DNA polymerase alpha catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus.

The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.     

Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity

Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.     

Structure and functional expression of cloned rat serotonin 5HT-2 receptor.

A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT-1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high-affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8-OH-DPAT as well as MDL-72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT-2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.     

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.     

Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA.

Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS-1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA.     

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.     

Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s

We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM4. The immunorelated polypeptide to P450 MC1 was not present in the control or the 3-methylcholanthrene-treated rat liver microsomes, whereas it was present in two human liver microsomal preparations. On the other hand, P450 MC4 was immunochemically related to rat P450d and rabbit LM4. The immunorelated polypeptide to P450 MC4 was present in the human and 3-methylcholanthrene-treated rat liver microsomes. We also isolated full-length cDNA clones encoding P450 MC1 and P450 MC4 mRNAs from a 3-methylcholanthrene-induced hamster liver cDNA library. The full-length cDNA clones of P450 MC1 and P450 MC4 contained 1771 and 1868 base pairs, which encoded polypeptides of 494 and 513 amino acids, respectively. RNA blot analysis revealed that the mRNAs for P450 MC1 and P450 MC4 were 2100 and 2600 bases in length, respectively. 3-Methylcholanthrene pretreatment increased the P450 MC1 mRNA level by 16-fold and the P450 MC4 mRNA level by 11-fold in the hamster livers. A comparison of the deduced amino acid sequences with other cytochrome P450s revealed that P450 MC1 was most similar to the mouse P450(15) alpha with 75% sequence identity, whereas P450 MC4 shared 87% identity with the rat P450d or mouse P3(450). These results indicated that P450 MC1 was a unique member (CYP2A8) in the P450IIA subfamily, whereas P450 MC4 was the hamster P450IA2.