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PubMed2003

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association.

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.

Bacterial Proteins↗
PubMed1997

Characterization of a KRAB family zinc finger gene, ZNF195, mapping to chromosome band 11p15.5.

We report the cDNA sequence of the zinc finger gene, ZNF195, which maps to chromosome 11p15.5. ZNF195 contains an N-terminal KRAB domain and 14 tandemly repeated Krüppel type zinc finger motifs at its C-terminus. Northern analysis shows expression of ZNF195 in adult heart, brain, placenta, skeletal muscle, and pancreas with a predominant transcript size of 4.3 kb. There is little expression in adult lung, liver, and kidney. In fetal lung, liver, kidney, and brain, the predominant transcript is 3.5 kb. Fetal brain also expresses a 4.3-kb transcript. RT-PCR analysis shows that two exons, 4a, which contains an inverted Alu sequence, and 4b, are differentially spliced and absent from the major transcript.

Adult↗
PubMed1981

Instability and reiteration of DNA sequences within the vaccinia virus genome.

The sequence arrangement within the nontranscribed portion of the inverted terminal repetition of the vaccinia virus genome exists in quasi-stable and unstable forms that are not distinguishable on the basis of viral infectivity. The unstable forms, which composed about 20% of a serially passaged stock of virus, were recognized by terminal heterogeneity on restriction endonuclease analysis. Instead of a single terminal fragment from each end of the genome, an array of eight or more fragments differing in size by 1650-base-pair increments was detected. This feature was not eliminated by repeated plaque purification, indicating that the population of DNA molecules with various numbers of reiterations can rapidly evolve from the DNA of a single virus particle. However, at each successive round of plaque purification, about 20% of the unstable isolates revert back to the more stable form. Stable forms are characterized by the presence of a set of 13-17 tandem 70-base-pair repeats on each side of a 435-base-pair intervening sequence near both ends of the genome. In contrast, the unstable forms possess sets of tandem repeats and intervening sequences that alternate many times in series. The transition between the two genomic forms and the evolution of the unstable form appear to be mediated by recombinational events.

Base Sequence↗
PubMed2004

DNA-binding specificity of AdpA, a transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus.

AdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and a DNA-binding domain with two helix-turn-helix motifs at its C-terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA-binding sequence, 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13-14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (-200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from -40 to -50 and from -50 to -60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13-14 bp and 2 bp. The DNA-binding domain is therefore connected to the ThiJ/PfpI/DJ-1-like dimerization domain with a flexible linker. The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.

Bacterial Proteins↗
PubMed1981

Site-specific inversion sequence of the herpes simplex virus genome: domain and structural features.

The genome of herpes simplex virus-1 consists of two covalently linked components, L and S, that invert relative to each other. The L and S components consist of unique DNA sequences bracketed by inverted repeats. The inverted repeats of the L component are designated ab and b' a' and those of the S component are designated a' c' and ca. The number of a sequences at the termini and at the L-S component junction varies from one to several copies. Insertion into the middle of the L component of a DNA fragment consisting of 156 base pairs (bp) of the b sequence, an entire a sequence of 501 bp, and 618 bp of the c sequence created a new site through which additional inversions in the genome occurred. Comparison of the nucleotide sequences of DNA fragments containing one and two a sequences defined the domain of the a sequence. The single a sequence consists of two 20-bp direct repeats (designated as DR1) bracketing a region that contains 19 tandem direct repeats of a 12-bp sequence (DR2) adjacent to three direct repeats of a 37-bp sequence (DR4), in addition to short stretches of unique sequences. The fragment with two tandem a sequences contained three copies of DR1-i.e., the intervening DR1 was shared by the two a sequences. Furthermore, one a sequence contained 22 copies of DR2 and two copies of DR4 whereas the second a sequence contained 19 copies of DR2 and two copies of DR4. These observations suggest that (i) amplification of the number of terminal and internal a sequences is the consequence of intramolecular or intermolecular recombination through DR1, (ii) the number of copies of DR2 and DR4 within the a sequence is not fixed and may vary as a consequence of unequal crossing over or slippage, and (iii) inversion results from intramolecular recombination between terminal and inverted a sequences.

Animals↗
PubMed1996

Cloning of Rous sarcoma virus enhancer factor genes. II. RSV-EF-II, abundantly expressed in fibroblasts and muscle tissue, binds to an octamer sequence, 5'-GTACCACC-3', in the noncoding strand of RSV enhancer.

Rous sarcoma virus (RSV) mainly replicates in avian fibroblasts, and the U3 enhancer region of the long terminal repeats of RSV contains the determinants for its tissue-tropic expression. We describe the cloning and characterization of an avian gene that encodes a protein capable of binding to the enhancer region of Rous sarcoma virus. A PCR-derived probe corresponding to the U3 region of RSV was used to isolate a cDNA clone by screening a chicken cDNA expression library. The cDNA is predicted to encode a polypeptide of 298 amino acids that is homologous to the Y-box (inverted CCAAT) family of DNA-binding transcription factors. This factor, which we refer to as Rous sarcoma virus enhancer factor-II (RSV-EF-II), shows 99% aa identity over a 105-amino-acid stretch that is highly conserved in all Y-box proteins, and is commonly referred to as the cold shock domain. RSV-EF-II selectively binds to single-stranded DNA, and the binding site, as determined by electrophoretic mobility shift assays, consists of the sequence 5' GTACCACC 3' located between nucleotides -112 to -119 in the noncoding strand of the RSV enhancer. Although RSV-EF-II shares considerable homology with the Y-box family of proteins, it does not bind to the inverted CCAAT boxes at positions -65 to -69 and -129 to -133 in the RSV LTR. Northern analysis indicates that RSV-EF-II-specific transcripts are expressed predominantly in avian fibroblasts and muscle tissue. The results of these binding and mRNA expression expriments suggest that RSV-EF-II may play an important role in tissue- and host-specific expression of RSV LTR-driven gene expression. Further, we show that RSV-EF-II acts as a repressor of transcription.

3T3 Cells↗
PubMed1992

Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (beta-subunit of RNA polymerase) and portions of rplL and rpoC.

The aphid Schizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola). The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment of B. aphidicola, homologous to the rplL-rpoB-rpoC portion of the Escherichia coli beta operon, was determined. The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (beta-subunit of RNA polymerase), and the first 209 amino acids of RpoC (beta'-subunit of RNA polymerase). The deduced sequences of B. aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins of E. coli. The sequences of two portions of the intergenic region between rplL and rpoB were nearly identical in both B. aphidicola and E. coli. One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB. A compilation of the codon usage for RpoB, RpoC, and other B. aphidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G + C) content of the DNA of this organism.

Amino Acid Sequence↗
PubMed1985

Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage.

The avian retroviral pol gene-encoded DNA endonuclease (pol-endo) has been shown to selectively cleave the viral long terminal repeat sequences (LTRs) in single-stranded DNA substrates in a region known to be joined to host DNA during integration (G. Duyk, J. Leis, M. Longiaru, and A.M. Skalka, Proc. Natl. Acad. Sci. USA 80:6745-6749, 1983). The preferred sites of cleavage were mapped to the unique U5/U3 junctions found only in covalently closed circular DNA molecules containing two tandem LTRs. The cuts occurred three nucleotides 5' to the axis of symmetry of the 12-of-15-base-pair nearly perfect inverted repeat which marks the LTR junction. Experiments with double-stranded supercoiled DNA substrates revealed a similar specificity for nicking. Also, the endonuclease associated with the pol cleavage product, pp32, has the same specificity as the alpha beta form. The limits of sequence required for site-selective cleavage near the U5/U3 junction were established with single-stranded DNA substrates. A domain no larger than 44 base pairs allowed site-selective cleavage in each strand in vitro. Recognition of either strand appeared to be independent of the other, and in each case, the critical sequence was asymmetrically distributed with respect to the U5/U3 junction. The predominant contribution was from the U5 domain; this is consistent with its conservation in the LTR sequences of a number of avian sarcoma and leukosis viruses.

Alpharetrovirus↗
PubMed1993

Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene.

Macrophage scavenger receptors (MSR) mediate the binding, internalization, and processing of a wide range of negatively charged macromolecules. Functional MSR are trimers of two C-terminally different subunits that contain six functional domains. We have cloned an 80-kilobase human MSR gene and localized it to band p22 on chromosome 8 by fluorescent in situ hybridization and by genetic linkage using three common restriction fragment length polymorphisms. The human MSR gene consists of 11 exons, and two types of mRNAs are generated by alternative splicing from exon 8 to either exon 9 (type II) or to exons 10 and 11 (type I). The promoter has a 23-base pair inverted repeat with homology to the T cell element. Exon 1 encodes the 5'-untranslated region followed by a 12-kilobase intron which separates the transcription initiation and the translation initiation sites. Exon 2 encodes a cytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, an alpha-helical coiled-coil, and exons 6-8, a collagen-like domain. The position of the gap in the coiled coil structure corresponds to the junction of exons 4 and 5. These results show that the human MSR gene consists of a mosaic of exons that encodes the functional domains. Furthermore, the specific arrangement of exons played a role in determining the structural characteristics of functional domains.

Amino Acid Sequence↗
PubMed1986

The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA.

We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.

Amino Acid Sequence↗
PubMed1997

A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats.

Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes). This recombinational rearrangement event is efficient and independent of RecA and RecBCD. In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats. In this model, the inverted repeats promote the formation of a DNA cruciform which is processed by an endonuclease into a linear DNA with two hairpin loops at its ends. Upon DNA replication, this linear dumbbell-like DNA is then converted to the inverted dimer. In support of this model, linear dumbbell DNA molecules with unidirectional origin of DNA replication (ColE1 ori ) have been constructed and shown to transform E.coli efficiently resulting in the formation of the inverted dimer. The ability of linear dumbbell DNA to transform E.coli suggests that the terminal loops may be important in bypassing the requirement of DNA supercoiling for initiation of replication of the ColE1 ori.

DNA↗
PubMed1997

The minute virus of mice (MVM) nonstructural protein NS1 induces nicking of MVM DNA at a unique site of the right-end telomere in both hairpin and duplex conformations in vitro.

The right-end telomere of replicative form (RF) DNA of the autonomous parvovirus minute virus of mice (MVM) consists of a sequence that is self-complementary except for a three nucleotide loop around the axis of symmetry and an interior bulge of three unpaired nucleotides on one strand (designated the right-end 'bubble'). This right-end inverted repeat can exist in the form of a folded-back strand (hairpin conformation) or in an extended form, base-paired to a copy strand (duplex conformation). We recently reported that the right-end telomere is processed in an A9 cell extract supplemented with the MVM nonstructural protein NS1. This processing is shown here to result from the NS1-dependent nicking of the complementary strand at a unique position 21 nt inboard of the folded-back genomic 5' end. DNA species terminating in duplex or hairpin configurations, or in a mutated structure that has lost the right-end bulge, are all cleaved in the presence of NS1, indicating that features distinguishing these structures are not prerequisites for nicking under the in vitro conditions tested. Cleavage of the hairpin structure is followed by strand-displacement synthesis, generating the right-end duplex conformation, while processing of the duplex structure leads to the release of free right-end telomeres. In the majority of molecules, displacement synthesis at the right terminus stops a few nucleotides before reaching the end of the template strand, possibly due to NS1 which is covalently bound to this end. A fraction of the right-end duplex product undergoes melting and re-folding into hairpin structures (formation of a 'rabbit-ear' structure).

Animals↗
PubMed1986

Activation of rat and human c-raf(-1) by rearrangement.

Activated c-raf(-1) gene was found in three transformants obtained by transfecting DNAs from rat hepatocellular carcinoma, metastasis of human colon cancer in mesocolon and normal mucosa from a different colon cancer patient. Rat and human activated c-raf(-1) genes were cloned into cosmid vectors; restriction enzyme mapping revealed both activated c-raf(-1) genes to have rearrangement in the center of the normal form of the gene, and the upstream sequences were replaced by unrelated sequences. Using genomic DNA fragments located immediately downstream of the recombination points, the activations of all these c-raf(-1) were shown to have occurred during the transfection process. The recombination points in both the rat and human clones isolated were located in the intron between exons 7 and 8, and nucleotide sequencing around these recombination points showed there to be an inverted repeat which could be involved in inducing in vitro recombination. Nucleotide sequencing of rat and human c-raf(-1) cDNAs revealed the upstream sequences, recombined to the 3' half of c-raf(-1), to be expressed as fusion mRNAs; the production of fused proteins was predicted from a long open reading frame, which is in-frame with the kinase domain encoded from the 3' half of the c-raf(-1) gene. There is a cysteine clustering region in an N-terminal region of the c-raf(-1) product deduced from the nucleotide sequence, and this cysteine clustering region was found to be highly homologous to that present in an N-terminal region of protein kinase C, although, in the latter cysteine clusters are present in duplicate. From analogy with the activation mechanism of protein kinase C, the N-terminal region of serine/threonine kinase coded by the c-raf(-1) gene is suggested to be a regulatory part of the enzyme activity, and it proposed that the replacement or truncation of this regulatory part could be the mechanism whereby c-raf(-1) is activated.

Amino Acid Sequence↗
PubMed2000

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980.

The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

Amino Acid Sequence↗
PubMed2006

Structure of the progesterone receptor-deoxyribonucleic acid complex: novel interactions required for binding to half-site response elements.

The DNA binding domain (DBD) of nuclear hormone receptors contains a highly conserved globular domain and a less conserved carboxyl-terminal extension (CTE). Despite previous observations that the CTEs of some classes of nuclear receptors are structured and interact with DNA outside of the hexanucleotide hormone response element (HRE), there has been no evidence for such a CTE among the steroid receptors. We have determined the structure of the progesterone receptor (PR)-DBD-CTE DNA complex at a resolution of 2.5 A, which revealed binding of the CTE to the minor groove flanking the HREs. Alanine substitutions of the interacting CTE residues reduced affinity for inverted repeat HREs separated by three nucleotides, and essentially abrogated binding to a single HRE. A highly compressed minor groove of the trinucleotide spacer and a novel dimerization interface were also observed. A PR binding site selection experiment revealed sequence preferences in the trinucleotide spacer and flanking DNA. These results, taken together, support the notion that sequences outside of the HREs influence the DNA binding affinity and specificity of steroid receptors.

Alanine↗
PubMed1993

Variability occurs in the inverted repeat region of genomic DNA from bovine herpesvirus 1 respiratory, genital and bovine herpesvirus 5 encephalitic isolates.

Restriction fragment length polymorphisms (RFLPs) were detected within BHV1.1, BHV1.2, and BHV5 genomes using the restriction enzyme PstI. The genomic areas of these changes has not been previously reported. Using Southern blot hybridization with DNA probes representing the entire genome of BHV1.1, areas of genomic variation were located for a respiratory isolate (BHV1.1), four vaccine isolates (BHV1.1), a genital isolate (BHV1.2), and two encephalitic isolates (BHV5). The most frequently observed RFLPs of BHV1.1 and BHV1.2 occurred within the internal repeat region and the left terminus of the unique long region. When two separate isolates of the encephalitic BHV5 were compared, RFLPs were detected in the internal and right terminal repeat regions. These are the regions of each genome from which immediate early genes are transcribed. No genomic variation was observed throughout the unique long and unique short regions for all BHV1 and 5 isolates examined.

Animals↗
PubMed1980

Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences.

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.

Animals↗
PubMed1993

A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity.

We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241. A number of random point mutations were constructed to test the validity of this assay. Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase. Purification of the Mtase proved to be difficult because the complex is weak. However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence. This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.

Amino Acid Sequence↗