SAAS bulletin, biochemistry and biotechnology最新文献

Eubacterium cellulosolvens 5494 is a cellulolytic gram positive bacterium isolated from the rumen. Substrate specific regulation has not been previously demonstrated in any members of this genera. However, we have recently found that E. cellulosolvens regulates some of its membrane proteins. Growth on different substrates, including cellulose and cellobiose, gave different SDS-PAGE profiles of proteins from the membrane fraction. Using scanning electron microscopy, we also found that growth of E. cellulosolvens on cellulose induces an ultrastructural complex that is not present when grown on any other substrate. Further study revealed that this ultrastructure was subsequently lost when an alternative substrate was made available to cellulose growing cells. We also found that cellulose, cellobiose, and maltose utilization are inhibited in the presence of a glucose analog, indicating glucose is the preferred substrate.

In eukaryotes, the guanine nucleotide exchange factor (eIF-2B) is a key protein in the control of polypeptide chain initiation. It catalyzes the exchange of chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the formation of a ternary complex (eIF-2.GTP.Met-tRNAf). The activity of eIF-2B is inhibited indirectly by phosphorylation of the smallest subunit of eIF-2 which sequesters eIF-2B into an inactive eIF-2(alpha P).eIF-2B complex. On the other hand, eIF-2B activity may be regulated directly by covalent modification of its largest subunit with different kinases, such as casein kinase (CK)-I, CK-II and glycogen synthase kinase (GSK)-3. After stimulation of mammalian cells by insulin or growth factors, the allosteric activation of eIF-2B activity by sugar phosphates and inositol phosphates may also provide an important parameter in the regulation of protein synthesis.

The small subunit of the ribosomal RNA has long been used as a tool in determining phylogenetic relationships. This project explored a region of the mitochondrial small subunit ribosomal RNA (MSrRNA) that is found only in the plant mitochondrial genome referred to as Variable Region 7 (V7). The V7 region of cauliflower and radish, both members of the Brassicaceae family, was amplified with the polymerase chain reaction, cloned into the expression vector PBSSK +, and sequenced. There was only a 0.3% sequences difference between the cauliflower and radish V7 region, thus suggesting that there will not be sequence variation in this region within a plant species. Cauliflower and radish V7 sequence was compared with the 6 other sequences of plant MSrRNA V7 region available: wheat, corn, oats, evening primrose, soybean and lupine. Based on percent difference between the V7 region sequences, a phylogenetic tree was constructed that supports the placement of these 8 species in Cronquist's morphologically based phylogenetic tree of flowering plants.

Sequence comparison of natural relaxins and the investigation of the structure function relationship of chemically synthesized relaxin analogs have been used to identify two arginine residues on the surface of the main helix of the B chain as hormone-receptor interaction site. This site is sensitive to structural changes, in particular the conformation of the A chain loop. Introducing the active site of relaxin into noncrossreacting structural analogs such as insulin and bombyxin required a four amino acid exchange. Both hybrid hormones bound to the anti-porcine relaxin antibody R6 with high affinity, and the insulin analog, with an additional C-terminal truncation of the B chain, crossreacted with rat relaxin-receptors.

Many human genes associated with disease have close homologs in yeast. Based on this homology, many human proteins have been studied using yeast expression systems. This paper will review research done in our laboratory using a yeast expression system to study the human protein galactose-1-phosphate uridylyltransferase, associated with galactosemia, as well as highlighting some of the advantages of this model system.

An enormous variety of metabolic processes are characterized by enzyme complexes, which are likely to play important roles in directing the efficient operation and specificity of cellular metabolism. In many cases membranes or cytoskeletal elements provide scaffolding for these highly ordered assemblies of enzymes. Biochemical and immunocytochemical studies indicate that the flavonoid biosynthetic pathway of higher plants involves a complex of sequentially-acting enzymes localized at the cytoplasmic face of the endoplasmic reticulum. This paper describes preliminary efforts to define the organization of this putative flavonoid biosynthetic complex and elucidate its role in controlling the synthesis of different flavonoid end-products in the model plant, Arabidopsis thaliana.

An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity. Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro. Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability. The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences. Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes. The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation. These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.

There is a chronic need to develop growth-enhanced fish for aquaculture. To meet this need we have developed techniques for genetically engineering fish to grow larger and faster. We found that the major difficulty in genetically engineering fish is the extremely high rate of mosaicism due to the late integration of transgenes into the genome. This delay also reduces the chances of passage of the transgene through the germ line. Consequently, we have engineered new vectors and mechanisms for accelerating the rate of integration of exogenous DNA into fish chromosomes.

Although most of the major discernible morphogenetic events in plants occur after germination, the overall architectural pattern of the mature plant is established during early events of embryogenesis. So far, few genes that are expressed specifically during embryogenesis have been identified. This is due primarily to technical difficulties associated with the mass ratios of the embryo and the surrounding maternal tissue and to the lack of molecular and cellular markers to direct screening efforts. We have developed a series of molecular approaches to study the early events of embryogenesis. These include 'virtual subtraction' of a cDNA library with high specific-activity cDNA probes generated from both seed and non-seed tissue, PCR amplification of gene family members from an immature seed cDNA library using primers specific to conserved domains, differential display analysis of mRNA populations and high throughput expressed sequence tag (EST) analysis. These techniques have led to the identification and isolation of several novel seed-specific cDNAs.