Formation of MboII vectors and cassettes using asymmetric MboII linkers

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

Primary structure of an aminoglycoside 6''-N-acetyltransferase AAC(6'')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase.

The gene aacA4 encoding an aminoglycoside 6'-N-acetyltransferase, AAC(6')-4, was cloned from a natural multiresistance plasmid, and its nucleotide sequence was determined. The gene was 600 base pairs (bp) long, and the AAC(6')-4 had a calculated molecular size of 22.4 kilodaltons and an isoelectric point of 5.35. The sequence of the 17 N-terminal amino acids was determined from the purified enzyme. The AAC(6')-4 gene was part of a resistance gene cluster, and its expression was under the control of the regulatory sequences of the beta-lactamase encoded by Tn3. The five N-terminal amino acids were identical to those of the signal peptide of the Tn3-encoded beta-lactamase, and the entire 5' region of aacA4, as far as it was sequenced (354 bp, including the promoter and the ribosome-binding site sequences), was identical to that of the beta-lactamase gene. This led us to presume an in vivo fusion between the beta-lactamase and the acetyltransferase genes. The latter was followed, in a polycistronic arrangement, by an aminoglycoside 3",9-adenylyltransferase gene, aadA, with an intergenic region of 68 bp. At a distance of ca. 1.3 kilobases in the 3' direction, we found remnants of a second Tn3-like element specifying an active beta-lactamase. At their 5' extremities, the two incomplete copies of Tn3, which were in tandem orientation, were interrupted within the resolvase gene. We speculate that Tn3-related sequences have played a role in the process of selection and dissemination of the AAC(6')-4 gene, which specifies resistance to amikacin and related aminoglycosides.     

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli . Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

蚯蚓纤溶酶基因(Efp-Ⅰ)在大肠杆菌中的克隆、表达及活性分析

从GenBank查询到的蚯蚓纤溶酶(earthworm fibrinolyti cenzyme,EFE)基因序列中,只有AY438624翻译的蛋白质序列与天然EfP-Ⅰ在N-端具有较高的相似性。根据该基因的5′与3′序列设计引物,通过RT-PCR从赤子爱胜蚓(Eisenia fetida)获得一个完整的基因(GenBank,DQ418454)。序列分析证明,由该基因编码蛋白质的N-末端与天然EfP-Ⅰ的N-末端的氨基酸顺序完全相同。ScanProsite prediction programs分析显示,该基因与AY438624相似性极高,二者均属于胰蛋白酶家族;不同的是,该基因编码蛋白质的序列中含有N-糖苷键的结构域,所以DQ418454是EfP-Ⅰ中的一个新基因。在此基础上,构建了该基因的原核表达载体pMAL-c2X-Efp-Ⅰ,并进行了转化、诱导和表达。Western blotting证明,表达产物同时具有MBP和EfP-Ⅰ的抗原特异性,是MBP和EfP-Ⅰ的融合蛋白(MBP-EfP-Ⅰ)。经亲和层析分离纯化的MBP-EfP-Ⅰ,在酪蛋白平板和纤维蛋白平板上表现出明显的纤溶酶活性。     

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.     

Determination of the recognition sites of cytosine DNA-methylases from Escherichia coli SK.

Two different cytosine DNA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5'...NpCpCpApGpGpN...3'. This sequence is identical with the recognition site of the hsp II type determined by RII plasmid but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5' side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GII site: 5'...NpGpGpApCpCpN...3'. In this case cytosine at the 3'-end of the recognition site is methylated.     

Site-specificity of DNA methylases from Shigella sonnei 47 cells

A complex approach involving isoplith analysis, enzymatic treatment of methylated isopliths and a computer analysis of experimental data has been used for determining site specificity of six methylases from Shigella sonnei 47 cells termed according to their specificity for a nitrous base and pI as MC4.2, MC5.3, MC6.2, MC7.4, MC8.4 and MA9.5. It has been found that the recognition site of MA9.5 is a palyndrome six-member structure of the 5'...GAATTC...3' type and that this enzyme is an isometimer with respect to MEcoRI. It has been demonstrated for the first time for methylases that the recognition site of MC4.2 is represented by a non-symmetrical four-member sequence, 5'...NCCCCN...3' characterized by unique blocking of cytosines. MC8.4 possesses a broad specificity of substrate recognition and methylates the cytosine residue within the composition of the non-symmetrical unique sequence 5'...N (C/Pu) CCN...3', whose 5'-terminal base is depleted in three nucleotides. MC5.3 methylates the 3'-terminal cytosine residue within the composition of the pentanucleotide palindrome recognition site, 5'...CCNGG...3'. MC6.2 and MC7.4 possess identical pentanucleotide recognition sites of 5'...(Py)CNG(Pu)...3', but are distinguished in pI. The latter finding has been shown for the first time for different methylases within one strain.     

Site-specific ribosomal DNA insertion elements in Anopheles gambiae and A. arabiensis: nucleotide sequence of gene-element boundaries.

The nucleotide sequence of the junctions between the 28S ribosomal gene and site-specific insertion elements from two sibling mosquito species, Anopheles gambiae and A. arabiensis, is reported. In both species, elements insert at the same point within the 28S gene, but this site is 634 basepairs (bp) 3' of the R1 (Type I) insertion site in Drosophila melanogaster. The two mosquito elements each have poly A tails and a polyadenylation signal, but the extreme 3' and 5' ends show no other similarity to each other or to any other insertion element. In both mosquito species, identical target site duplications of 17 bp are generated. The sequence TNTCCCTNT found in this duplication is also found in the 14 bp target site duplications that flank R1 elements in D. melanogaster. Another sequence in this duplication, GGGATAACT, is very similar to the sequence GGGAGTAACT found in the 24 base sequence required by the Bombyx mori R2 endonuclease.     

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes.     

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.     

The role of branchpoint-3'' splice site spacing and interaction between intron terminal nucleotides in 3'' splice site selection in Saccharomyces cerevisiae.

A conserved 3' splice site YAG is essential for the second step of pre-mRNA splicing but no trans-acting factor recognizing this sequence has been found. A direct, non-Watson-Crick interaction between the intron terminal nucleotides was suggested to affect YAG selection. The mechanism of YAG recognition was proposed to involve 5' to 3' scanning originating from the branchpoint or the polypyrimidine tract. We have constructed a yeast intron harbouring two closely spaced 3' splice sites. Preferential selection of a wild-type site over mutant ones indicated that the two sites are competing. For two identical sequences, the proximal site is selected. As previously observed, an A at the first intron nucleotide spliced most efficiently with a 3' splice site UAC. In this context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU. Our data indicate that the 3' splice site is not primarily selected through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the branchpoint but does not occur by a simple leaky scanning mechanism.     

Expression and base sequence of the citrate synthase gene of Acinetobacter anitratum

The sequence of 1895 base pairs of Acinetobacter anitratum genomic DNA, containing the structural gene for the allosteric citrate synthase of that Gram-negative bacterium, is presented. The sequence contains an open reading frame of 424 codons, the 5' end of which is the same as the N-terminal sequence of A. anitratum citrate synthase, less the initiator methionine. The inferred amino acid sequence of the enzyme is about 70% identical with that of citrate synthase from Escherichia coli, which like the A. anitratum enzyme is sensitive to allosteric inhibition by NADH. There is also a more distant homology with the nonallosteric citrate synthases of pig heart and yeast. The gene contains sequences that strongly resemble those found in E. coli promoters, an E. coli type of ribosomal binding site, and a hyphenated dyad sequence at the 3' end of the gene which resembles the rho-independent terminators found in some E. coli genes. The plasmid clone containing the A. anitratum citrate synthase gene pLJD1, originally isolated because it hybridized with the cloned E. coli citrate synthase gene under conditions of reduced stringency, produces large amounts of A. anitratum citrate synthase in an E. coli host which lacks citrate synthase. This work completes proof of the hypothesis that the three major kinds of citrate synthases are formed of similar subunits, although their functional properties are different.     

DNA binding and recognition by the IIs restriction endonuclease MboII

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.     

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.