Journal of molecular and applied genetics最新文献

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.

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.

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.

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.

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.

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).

Mutants with deletions in the control region of simian virus 40 (SV40) were tested for their ability to direct late transcription in a nuclear extract derived from HeLa cells. Primer extension analysis revealed that late SV40 transcription initiates predominantly at two sites in vitro, one of which corresponds to the major in vivo start site at nucleotide 325, while the other site is located at nucleotide 170. A series of 5',3', and internal deletions of the putative promoter region were used to define two distinct control elements that appear to function independently of each other and that are located upstream from each of the in vitro initiation sites. In addition, transcription from the initiation site at 325 is also influenced by GC-rich sequences (CCGCCC) found within a regulatory region that consists of two 21 bp perfect repeats and a third degenerate repeat located 250 bp upstream from the major late initiation site. These six upstream GC blocks, which lie directly upstream from the initiation site at 170, also affect transcription from this start site. Although these 21 bp repeats are known to be an important part of the SV40 early promoter, our findings suggest that they are also involved in modulating the levels of late transcription in vitro.

Recombinant bacteriophages containing the alcohol dehydrogenase (ADH) gene from Drosophila affinidisjuncta have been isolated by virtue of their cross-hybridization to the previously cloned ADH gene from D. melanogaster. Within the 17 kilobases of cloned DNA represented in the phage genomes, the sequences hybridizing to the D. melanogaster ADH gene lie roughly in the center. The only region of detectable hybridization to cDNA made from templates of D. affinidisjuncta larval poly(A)-containing RNA maps to the same portion of the cloned DNA. Verification that the phages carry the ADH structural gene was obtained by hybrid-selecting ADH mRNA, translating it in vitro, and immunoprecipitating the resulting ADH polypeptide. Analysis of genomic DNA suggests that the ADH gene and most flanking sequences are present only once in the haploid genome. However, 3' to the ADH gene, two separable repetitive elements are found. Both repetitive elements are probably small and poorly conserved in the genome, and neither interferes with localization of the ADH gene, by in situ hybridization, to a position near the base of the third chromosome. Analysis of ADH transcripts demonstrates that there are at least four RNAs produced by the ADH gene. Two size classes of RNA are seen at each stage of development. In addition, ADH transcripts from larvae and adults differ from one another in a reproducible manner.

An approximately 1.4 kb fragment of DNA called Mu1, mutationally inserted into the Adh1 locus of maize in a Robertson's Mutator plant, has been cloned. The instability of the mutation induced by this element, the nature of the Robertson's Mutator system, and terminal inverted repeats indicate that the 1.4 kb insert is a transposable element. All Robertson's Mutator corn lines have Mu1-like elements, at copy numbers of 10-70 per diploid genome. The basic size of these multiple interspersed copies is generally the same. The elements are found on different genomic restriction fragments in closely related individuals, indicating a high degree of mobility. Aside from the one corn line identified by Robertson in the mid-1970s as Mutator, all maize lines tested, plus several near and remote corn relatives, have no detectable DNA which cross-hybridizes strongly with Mu1.