We have identified cloned fragments of the soybean genome that hybridize to total soybean tRNA. Five of these clones, chosen at random, have unique patterns of restriction endonuclease sites and contain only a small region that is homologous to tRNA (less than 1 kb of 10-12 kb cloned). Two of the hybridizing fragments were subcloned and regions of about 600 bp including the homologies were sequenced. Each region contains a single putative tRNA gene, for tRNAasp or tRNAmet, surrounded by DNA rich in AT basepairs (68-82%). Neither sequence encodes the amino acid-accepting -CCA terminus. The tRNAasp gene does not contain any intervening sequences, but the tRNAmet gene has an 11-bp sequence in the anticodon loop that would not be expected in the mature tRNA. There appear to be a small number of sequences within the soybean genome that share homology with each of the regions containing a putative tRNA gene.
Two beta globin gene alleles have been cloned and characterized from a patient with beta + thalassemia. Both beta genes have single base mutations in the small intervening sequence (IVS 1); one 6 nucleotides and the other 110 nucleotides from the 5' end of IVS 1. Both genes lead to abnormal splicing of beta globin mRNA precursors when expressed in HeLa cells. Despite the fact that both alleles produce some normal beta globin mRNA transcripts, the patient has clinically severe beta + thalassemia (Cooley's anemia).
A 213-bp region of noncoding DNA upstream of the ATG start codon of the Pseudomonas carboxypeptidase G2 (CPG2) structural gene has been shown to contain the CPG2 promoter. The mRNA start point (+1) on the DNA sequence has been identified by mapping the 5' end of the CPG2 transcript. The identified promoter region contains a -10 region (TATAAG) that closely resembles the Escherichia coli consensus sequence (TATAAT), but has no easily recognisable -35 region. The lack of homology in the -35 region explains why this particular pseudomonad gene is poorly expressed in E. coli. Similar sequence differences may turn out to be the cause of the generally observed inefficient expression of Pseudomonas genes in E. coli. The promoter region also carries a sequence (CTGGCACTCGAATTGCT) that closely matches the consensus nif promoter sequence (CTGGPyAPyPuNNNNTTGCA) of Klebsiella pneumoniae and Rhizobium, and a similar sequence (ATGGCATGGCGGTTGCT) found in the promoter region of the xylABC operon of the TOL plasmid of Pseudomonas putida.
A facile immunoaffinity chromatography method is described for the purification of lacZ fusion gene products. The method is general for any molecule antigenically related to beta-galactosidase and involves only a single step. We report its use to purify the products of lacZ fusions with a bacteriophage gene, phi X174E, and an Escherichia coli chromosomal gene, prlA. The hybrid protein products of both of these genes are membrane bound and present in very low molar amounts with respect to total cellular protein. Evidence is presented that substrate-affinity chromatography is not applicable to the isolation of low-level fusion proteins such as these.
We have sequenced a genomic subclone (pLg AB19/H5c) of Lemna gibba nuclear DNA containing a complete chlorophyll a/b protein coding region and 5' and 3' flanking nucleotides. The coding region contains an intron of 84 nucleotides that has features characteristic of a transposable element. Evidence from S1 nuclease mapping experiments is consistent with correct transcription and splicing of the AB19 or another closely related intron-containing gene. The encoded precursor polypeptide of 264 amino acid residues has a predicted Mr of 28,327. Approximately 35 N-terminal residues are cleaved from this protein to form the mature apoprotein. We have used theoretical considerations of protein structure to propose an experimentally testable model of the structure of this protein in thylakoid membranes.
Transient expression of an SV40-based shuttle vector similar in design to commonly used vectors is shown to be inefficient when compared to expression of SV40 viral DNA. To eliminate this problem, we have designed and constructed a new vector, pSVPiC, which contains a minimal noninterfering plasmid component, the 885-bp plasmid PiAN7, and two SV40 promoter/origin regions. Transient expression of the SV40 late region from pSVPiC is much more efficient than that from previously used vectors and even more efficient than that from SV40 viral DNA. When the gene for rabbit beta-globin is placed in the late region of pSVPiC, it is also expressed at high levels, indicating that this shuttle vector is generally useful for expressing eukaryotic genes.
Penicillin acylase is processed from a 90-kD precursor through the cleavage of a leader peptide and two further endopeptidase cleavages to yield an enzyme that contains a 22-kD (or 23-kD) and a 65-kD subunit. The endopeptidase cleavages require an intact carboxy terminus. This type of processing appears to be unique for a prokaryotic enzyme, having its most closely related analog in the synthesis and processing of preproinsulin and other eukaryotic hormones.
We have previously reported that the promoter of the thymidine kinase (TK) gene of herpes virus (HSV) can be replaced with the early promoter of SV40. In the present study we report the construction of a TK- plasmid (TK-pML-BglII) in which the TK promoter has been removed and a new BglII site has been regenerated for the insertion of exogenous promoters in front of this nonexpressed gene. We have tested the feasibility of using this plasmid for the isolation of unknown promoters by inserting DNA fragments containing the early and late promoters of human papovaviruses and have obtained activity. We have also inserted the fragments containing the 72-bp repeat enhancer sequences of SV40 and 107-bp repeat of the human BK virus into the intact TK plasmid (TK-pML) and observe increased frequency of transformation of mouse Ltk- cells to the TK+ phenotype. These results show that the TK system can be used as a vehicle for the isolation of unknown mammalian promoters and/or enhancers.
Phytohemagglutinin (PHA), the major lectin of the common bean Phaseolus vulgaris, is synthesized during the development of the seeds. In most cultivars PHA makes up 5-10% of the total seed protein, but certain cultivars do not contain PHA. In vivo labeling of a normal cultivar (Greensleeves) and a PHA-minus cultivar (Pinto 111) showed that PHA was not synthesized in the PHA-minus cultivar. To find out whether the lack of synthesis was due to the absence of mRNA for PHA, recombinant cDNA clones for PHA were obtained. Total poly(A)+ RNA was isolated from cotyledons of developing seeds of Greensleeves and used to direct cDNA synthesis. The double stranded cDNA was cloned in pUC8 and transformants of Escherichia coli screened with pPVL134, a recombinant plasmid which contains the complete coding sequence for a PHA-like protein. Two weakly hybridizing clones (pSC1 and pSC2) were selected. Hybrid selection experiments showed that these two clones selected mRNAs which could be translated into polypeptides identical in size to PHA and recognized by antibodies to PHA. The recombinant pPVL134 selected mRNA which translated into polypeptides which were slightly smaller than those of PHA, and poorly recognized by antibodies to PHA. The recombinant clones were used to demonstrate that the genes for PHA and for the PHA-like protein are under temporal control during seed development. The cultivar Pinto 111, which has no detectable PHA, also has greatly reduced levels of mRNA for PHA. However, the gene for the PHA-like protein encoded by pPVL134 is expressed to the same degree in the cultivars Greensleeves and Pinto 111.
In several species of Rhizobium the three genes which encode the nitrogenase complex are separated into two operons, nifH and nifDK. We have mapped the transcriptional promoter sites for these two operons from R. japonicum USDA strain 110 by S1 protection analyses using bacterial RNA isolated from soybean nodules. Transcription of the nifDK operon is initiated at a site located 46 nucleotides upstream of the proposed translation initiation codon. nifH transcription initiates predominantly at a site 152 nucleotides upstream of the proposed translation initiation codon. An additional minor start for nifH is found 35 nucleotides downstream of the major initiation site. The nucleotide sequences of these promoter sites are presented. Comparison of the major R. japonicum nif promoter sequences reveals a high degree of sequence homology with the conserved sequences clustered in the -11 to -15, -21 to -25, -27 to -31, and -38 to -41 regions. Conservation is also observed for the -11 to -15 and -21 to -25 regions with the R. meliloti nifHDK, a cowpea Rhizobium nifH, and even Klebsiella pneumoniae nif promoters. The -27 to -31 and -38 to -42 region homologies are not conserved with the other known nif promoters. These results are discussed in light of models for nif promoter activation.
Normal adult ducks possess two main types of hemoglobin: a major species (80%), HbI (alpha I2 beta I2), and a minor species (20%), HbII (alpha II2 beta II2). We have cloned recombinant cDNAs for the duck globin mRNAs using the ribosubstitution floppy loop technique. We present here the sequence for a duck alpha globin mRNA closely related to the chicken alpha D globin mRNA. Our new sequence data also include the 5' noncoding regions of the duck alpha A, alpha D, and beta globin mRNAs. Analysis of the untranslated regions of these mRNAs reveals several conserved sequences which may be important in the regulation of gene expression.
A naturally occurring variety of soybean Glycine max cv. Keburi, which lacks the alpha'-subunits of the 7S seed storage protein (beta-conglycinin), was recently described by Kitamura and Kaizuma. Keburi beta-conglycinins contain a normal complement of alpha-, beta-, and gamma-subunits that accumulate in a temporally and spatially regulated manner comparable to that of the standard cultivar Provar. Poly(A)+ RNA isolated from mid-to-late maturation stage Keburi seeds was translated in vitro, yielding products equivalent to those of Provar poly(A)+ RNA except that Keburi mRNA did not produce the pre-alpha'-subunit polypeptide. The basis of the Keburi phenotype was determined by examining genomic DNA using Southern blot hybridization. Restriction fragments isolated from a cloned 11.5 kb EcoRI fragment of genomic DNA containing a normal alpha'-subunit gene (Gmg 17.1) were used as hybridization probes. Sequences far upstream of the alpha'-subunit gene were present in both Provar and Keburi cultivars. However, hybridization reactions with probes from within the gene demonstrated that a deletion had occurred in Keburi DNA beginning immediately 5' to the alpha'-subunit gene represented on this 11.5 kb fragment. The deletion extends through most of the coding sequences, producing the Keburi phenotype.
In an effort to determine if particular regions of the cauliflower mosaic virus (CaMV) genome could be associated with particular phenotypic characters, strains of CaMV differing markedly in biological properties were recombined to produce hybrids. DNA from pairs of (infectious) genomic clones was cleaved with restriction endonucleases, then mixed and ligated. Recombinants were found by screening transformants in E. coli, or by selection in vivo for infectious hybrids. Recombinants in infected turnip plants were characterized by restriction endonuclease mapping of their DNA to confirm the hybrid genotype. New hybrid strains that induced less severe disease, or conversely, more severe disease than either parent were observed. The experiments revealed that typical disease expression, consisting of leaf chlorosis and mottling, mapped to a genome segment containing open reading frame VI (ORF VI) and the full-length promoter. This basic disease symptom was found to be influenced by other regions of the genome. Insect transmissibility mapped to ORF II. The ability to develop generalized infections in solanaceous plants was tested in hybrids between CaMV CM1841 and a variant that infects Datura stramonium systemically. In this case the systemic mobilization of virus appeared to be controlled by ORF VI, suggesting that this gene may function in cell-to-cell movement of virus.
The DNA sequence has been determined for a 3.8-kilobase region which encodes the mercury resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals four open reading frames which could encode proteins of 12,391, 9,429, 14,965, and 58,781 daltons. On the basis of their sizes, amino acid compositions, hydropathicities, and estimated isoelectric points, the peptides encoded by these open reading frames correspond to the four previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The NR1 mer locus is 63.4% GC overall, and the Hg(II) reductase protein sequence is 90% homologous to that of Tn501. The region encoding the merR (positive regulatory) function has three open reading frames. The smallest of these possible merR peptides (6,457 daltons) begins approximately 280 bp to the right of the adjacent IS1b and reads towards the structural genes of the mer operon. The next largest reading frame (13,139 daltons) in the merR region begins 37 bp to the left of the beginning of the smallest peptide and also reads towards the structural genes. The largest reading frame (15,907 daltons) in the merR region lies on the complementary strand and reads away from the structural genes towards IS1b. Although attempts to visualize the merR gene product were not successful, in vitro mutagenesis allows us to eliminate the largest reading frame as a merR candidate. We were also able to show that approximately 50% of the smallest detectable mer peptide (9,429 daltons) is located in the periplasm.
Several alpha-amylase clones were identified by screening a group of 345 cDNA clones derived from poly(A)+RNA from gibberellic acid (GA)-treated barley aleurone layer cells using an alpha-amylase cDNA clone that we had characterized previously as the hybridization probe. The alpha-amylase clones showed differences in the distribution of restriction enzyme sites, in the extent of cross hybridization, and in nucleotide sequence. There are at least three types of alpha-amylase clones in our collection, and the alpha-amylase cDNA clone reported by Rogers and Milliman [J. Biol. Chem. (1983) 258: 8169-8174] is a fourth type. The accumulation of alpha-amylase mRNAs in aleurones as a function of time after GA addition and of hormone concentration in the incubation medium was studied using different alpha-amylase cDNA clone probes. These studies indicate a differential control of the expression of the alpha-amylase genes by GA.
The nucleotide sequence of a tumor morphology gene, tmr, from the Agrobacterium tumefaciens Ti-plasmid, pTiA6NC, and its flanking 5' region was determined by M13 "dideoxy" procedures. The DNA sequence reveals an open reading frame capable of encoding a 240 amino acid protein. We have identified the polyadenylated transcript initiation and termination sits by S1 nuclease mapping. The extent of the sequence required for transcription 5' to the start of transcription has been delimited by two transposon insertions. The first of these maps at -- 121 with respect to transcription initiation and results in the wild-type phenotype, the second insertion maps at about -85 and results in a tmr phenotype.
The expression of a protein-coding DNA fragment in a microorganism such as Escherichia coli requires that the exogenous DNA segment be inserted precisely in phase with bacterial translation initiation signals. We report the construction of derivatives of the M13 vectors M13mp7 and M13mp701 in which a HindIII site has been inserted, within the N-terminal section of the beta-galactosidase gene, in all three phases of translation. These vectors may thus be used for the expression, under the control of the lac promoter and translation initiation signals, of protein coding DNA segments flanked by a HindIII site. In all cases the insertion of a DNA fragment can be recognized by the abolition of beta-galactosidase activity. These vectors have been used to direct the expression, in E. coli, of a cDNA segment coding for the rabies virus surface glycoprotein. The proteins produced have been shown to react with antisera raised against authentic rabies glycoprotein.
In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R. meliloti nif-genes. Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination. Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins. The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype. Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence. Gene products were identified by immunoprecipitation with specific antisera. Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E. coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells. The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.
Misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate was studied using 32P-labeled DNA primers annealed to the appropriate template DNA, and polyacrylamide-urea gel electrophoresis to measure the extension of the primer chains. With most primer-template combinations, greater than 50% of the primers were extended by the addition of a single incorrect nucleotide onto the end of the primer chain. Unexpectedly, one primer-template combination was not extended in the presence of dCTP, although misincorporation occurred with the other deoxyribonucleoside triphosphates. In another case, terminal misincorporation of two rather than one dT residue was observed. The primer termini containing unpaired nucleotides were efficiently extended upon addition of the other three deoxyribonucleoside triphosphates, even in the case where the primer terminus contained two unpaired nucleotides. Misincorporation was confirmed by direct sequence analysis. These results indicate that the frequency of mutations following misincorporation by reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate should be sufficiently high to allow detection of mutants by simple screening procedures. An analysis of the sequence of a mutant resulting from misincorporation at the M13mp2 AvaII site revealed that following introduction of the DNA into Escherichia coli cells, mismatch repair preceded replication.
Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.
