SAAS bulletin, biochemistry and biotechnology最新文献

We are developing an in vitro system for poliovirus RNA recombination. In this system, two mutant RNAs are replicated with poliovirus RNA-dependent RNA polymerase. Recombination will produce RNAs containing neither mutation and will be the only progeny RNAs that are infectious. We will use this system to determine what proteins and reaction conditions are required for recombination and to study the details of the mechanism of recombination.

Cytokines are a family of glycoproteins produced by various cell types in response to specific stimuli that regulate the immune response. This paper reviews recent studies on two different cytokines, each with its own effector cell type: interleukin-2 (IL-2), which regulates lymphoid cell responses; and granulocyte colony-stimulating factor (GCSF), which regulates neutrophil responses. In the first study, administration of IL-2 to the bovine mammary gland was found to stimulate the expansion of lymphocyte populations and increase local antibody production. In the second study, systemic administration of GCSF increased peripheral blood as well as milk neutrophil populations, which afforded some protection against Staphylococcus aureus challenge. Results suggest a role for cytokines in the control of mastitis in dairy cattle.

RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.

The recombinant DNA junctions at which pUB110 and Bacillus subtilis chromosomal DNA were joined to form the plasmid pKBT1 were cloned and sequenced. From the sequencing data we conclude that the pUB110 sequence is intact in the pair of cloned pKBT1 fragments and pTL12 sequences are not present. A molecular model for the formation of pKBT1 based on structural motifs characteristic of the joint sites is presented.

The current generally accepted model of eukaryotic gene regulation is essentially a simple one. Regulatory proteins containing separable DNA binding and transcriptional activation domains, bind to specific DNA sequences in promotors and interact directly or indirectly with the TATA Box binding factor to increase the rate of transcription initiation at selected promotors. Here we present observations suggesting that the process may be more complex.

The flagellar filaments of bacteria of the genus Salmonella are highly immunopotent and antigenically diverse. It is proposed to develop vaccines by replacing the flagella of live attenuated Salmonella strains with engineered flagellar filament proteins containing foreign epitopes of medical and agricultural importance. As an initial step in this process, the major linear epitope regions of one filament protein have been identified.

Acinetobacter calcoaceticus is a metabolically versatile microorganism that is naturally competent for DNA uptake and incorporation. We have exploited the natural state of competency for studies involving the cloning, organization and expression of genes encoding catabolic enzymes. A. calcoaceticus is able to take up, at high efficiency, genetically engineered DNA, incorporate the DNA and stably maintain and express the DNA. Sequence analysis of cloned A. calcoaceticus DNA reveals a great deal of internal repetition and secondary structure, but no specific sequences associated with uptake appear to be present. Uptake and transformation occurs in solid and liquid medium, at a wide range of DNA concentrations and with little restriction barrier to the source of the transforming DNA.

Cooperative interactions between regulatory proteins and RNA polymerase are a common feature of transcriptional systems. We have developed a method, based on the electrophoresis mobility shift assay, for the measurement of cooperative effects in the binding of proteins to DNA restriction fragments. Using this approach we have identified a hitherto unknown interaction between the E. coli lactose repressor and CAP proteins. We suggest that this interaction plays a role in the control of the lactose operon that is not predicted by current regulatory models.

The larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is expressed at a very high level in the fat body of third-instar larvae. Here we report that Lsp-2 transcription in adult flies produces a unique mRNA localized in the adult adipose tissue of the head in both sexes. To identify regulatory regions of this Drosophila gene, Lsp-2 5'-flanking DNA sequences were fused to the E. coli beta-galactosidase gene (lacZ). Transient expression of the hybrid gene in third-instar larvae indicates that 230 bp just upstream from the 'TATA box' of the Lsp-2 gene are sufficient for larval fat body-specific expression.

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E.coli, isolated from E.coli cell extracts, and identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control region (ICR) of the Xenopus 5S ribosomal RNA gene from DNase I digestion. Intact protein bound specifically to the entire ICR (+96 to +43). One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 protected the 5S gene ICR from positions +96 to +63. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR.