牛凝乳酶原基因在大肠杆菌中表达调控的研究

Shine-Dalgarno序列与起始密码子之问的距离与组成对凝乳酶原基因表达有明显的影响,可导致其表达水平有15倍之差。SD序列至ATG之间为15bp不利于表达,表达质粒中sD-ATG在7-11bp之间都有可能获得高效表达;但决定因素不是简单的长度,而是RBS附近可能的二级结构即△G的大小、SD序列及ATG中参与配对的碱基数目。将pTLC23中凝乳酶原cDNA3＇端端非翻译区插入终止密码子TGA与转录终止子rrnBT₁T₂之间适当位置可提高凝乳酶原基因的表达,这可能是因为这段序列能形成由53个碱基对和8个碱基组成的稳定的mRNA二级结构,起到转录终止子的作用,而一般认为串联终止子对终止转录更为有效。     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus

The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine-Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.     

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.     

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.     

Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides.

Nuclear factor I (NFI) binds tightly to DNA containing the consensus sequence TGG(N)6-7GCCAA. To study the role of the spacing between the TGG and GCCAA motifs, oligonucleotides homologous to the NFI binding site FIB-2 were synthesized and used for binding assays in vitro. The wild-type site (FIB-2.6) has a 6bp spacer region and binds tightly to NFI. When the size of this spacer was altered by +/- 1 or 2bp the binding to NFI was abolished. To further assess the role of the spacer and bases flanking the motifs, two oligonucleotide libraries were synthesized. Each member of these libraries had intact TGG and GCCAA motifs, but the sequence of the spacer and the 3bp next to each motif was degenerate. The library with a 6bp spacer bound to NFI to 40-50% the level of FIB-2.6. The library with a 7bp spacer bound to NFI to only 4% the level of FIB-2.6 and some of this binding was weaker than that of FIB-2.6 DNA. This novel use of degenerate DNA libraries has shown that: 1) the structural requirements for FIB sites with a 7bp spacer are more stringent than for sites with a 6bp spacer and 2) a limited number of DNA structural features can prevent the binding of NFI to sites with intact motifs and a 6bp spacer region.     

Deletions/insertions, short inverted repeats, sequences resembling att-lambda, and frame shift mutated open reading frames are involved in chloroplast DNA differences in the genus Oenothera subsection Munzia

Summary A restriction fragment length mutation has been mapped in the large single copy region of the chloroplast DNA from two Munzi-Oenothera species. Fragments containing the deletion/insertion were cloned, further analysed by additional restriction enzymes, and sequenced. A deleted/inserted 136 bp sequence was identified upstream of the 5 end of a tRNA-Leu (UAA) gene and presumably is located in the spacer between this gene and a tRNA-Thr (UGU) gene. The endpoints of the 136 bp sequence are covered by short inverted repeats. Complementary inverted repeats are present in the middle of the deleted/inserted sequence. The repeats are part of sequences resembling the lambda chromosomal attachment site (att-lambda) which is essential for site specific recombination in the lambda/ Escherichia coli system. Possible interactions of the repeats during the deletion/insertion process are discussed. The spacer also contains a 1 bp deletion/insertion within an open reading frame (ORF). Due to this frame shift mutation the ORF sizes are quite different between the two Oenothera species.     

Nucleotide sequence of the Clostridium thermocellum laminarinase gene.

The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions. The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661. The ORF startswith the translation initiation codon ATG. This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT). A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa). The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).     

The role of the loxP spacer region in P1 site-specific recombination.

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.     

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.     

Site-specific DNA binding of nuclear factor I: effect of the spacer region.

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus type 2 DNA in vitro and in vivo. To study sequence requirements for the interaction of NFI with DNA, we have measured the binding of the protein to a variety of synthetic sites. Binding sites for NFI (FIB sites) were previously shown to contain a consensus sequence composed of 2 motifs, TGG (Motif 1), and GCCAA (Motif 2), separated by a 6 or 7bp spacer region. To assess conserved sequences in the spacer region and flanking sequences which affect NFI binding, we have isolated clones from oligonucleotide libraries that contain the two motifs flanked by 3 degenerate nucleotides and separated by degenerate spacer regions of 6 or 7 nucleotides. With a 6bp spacer region, a strong bias exists for a C or A residue in the first position of the spacer. Sites with a 7bp spacer region contain a G and C or A residue at the first and second positions, respectively, of the spacer, but also possess conserved residues at other positions of the site.