Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Genetic relatedness of human DNA polymerase beta and terminal deoxynucleotidyltransferase

The Protein Identification Resource (PIR) protein sequence data bank was searched for sequence similarity between known proteins and human DNA polymerase beta (Pol beta) or human terminal deoxynucleotidyltransferase (TdT). Pol beta and TdT were found to exhibit amino acid sequence similarity only with each other and not with any other of the 4750 entries in release 12.0 of the PIR data bank. Optimal amino acid sequence alignment of the entire 39-kDa Pol beta polypeptide with the C-terminal two thirds of TdT revealed 24% identical aa residues and 21% conservative aa substitutions. The Monte Carlo score of 12.6 for the entire aligned sequences indicates highly significant aa sequence homology. The hydropathicity profiles of the aligned aa sequences were remarkably similar throughout, suggesting structural similarity of the polypeptides. The most significant regions of homology are aa residues 39-224 and 311-333 of Pol beta vs. aa residues 191-374 and 484-506 of TdT. In addition, weaker homology was seen between a large portion of the 'nonessential' N-terminal end of TdT (aa residues 33-130) and the first region of strong homology between the two proteins (aa residues 31-128 of Pol beta and aa residues 183-280 of TdT), suggestive of genetic duplication within the ancestral gene. On the basis of nucleotide differences between conserved regions of Pol beta and TdT genes (aligned according to optimally aligned aa sequences) it was estimated that Pol beta and TdT diverged on the order of 250 million years ago, corresponding roughly to a time before radiation of mammals and birds.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations

Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal antibodies specific for this protein. Thus, we have N-formylated the single tryptophan found in horse cytochrome c at position 59 and N-carbethoxylated one of the histidyl residues, which was determined to be at position 26 by the analysis of proteolytic cleavage fragments of the modified protein using liquid secondary ion-mass spectrometry on triple quadropole or tandem quadropole Fourier transform instruments. We discuss the impact of these modifications on the antigenicity of horse cytochrome c with regard to the conformational perturbations introduced by such modifications and with reference to our previous studies on the binding sites of these antibodies using other methodologies (Jemmerson, R., and Paterson, Y. (1986) BioTechniques 4, 18-31).     

Nucleotide sequence of a fragment of the rat tryptophan oxygenase gene showing high affinity to glucocorticoid receptor in vitro.

A nucleotide sequence (3.2 kb) of a DNA region located approximately between introns 4 and 7 of the rat tryptophan oxygenase gene was determined. Using filter binding studies and monoclonal antibodies against the glucocorticoid receptor a high affinity binding of this region to the glucocorticoid receptor was demonstrated.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases

A cDNA encoding DNA (cytosine-5)-methyltransferase (DNA MeTase) of mouse cells has been cloned and sequenced. The nucleotide sequence contains an open reading frame sufficient to encode a polypeptide of 1573 amino acid residues, which is close to the apparent size of the largest species of DNA MeTase found in mouse cells. The carboxylterminal 570 amino acid residues of the inferred protein sequence shows striking similarities to bacterial type II DNA cytosine methyltransferases and appears to represent a catalytic methyltransferase domain. The amino-terminal portion of the molecule may be involved in regulating the activity of the carboxyl-terminal methyltransferase domain, since antibodies directed against a peptide sequence located within this region inhibits transmethylase activity in vitro. A 5200 base DNA MeTase-specific mRNA was found to be expressed in all mouse cell types tested, and cell lines known to have different genomic methylation patterns were found to contain DNA MeTase proteins of similar or identical sizes and de novo sequence specificities. The implications of these findings for an understanding of the mechanisms involved in the establishment and maintenance of methylation patterns are discussed.     

Mapping epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum using fusion proteins.

Epitopes for a number of monoclonal antibodies (mAbs) binding (Ca(2+)-Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum have been defined by studying binding to fusion proteins generated from cDNA fragment libraries. Comparison of these results with those of previous studies of binding of mAbs to proteolytic fragments of the ATPase have allowed the definition of the epitopes to within approx. 100 residues and for one (mAb 1/2H7) to within 45 residues. The experiments suggest considerable exposure of the nucleotide binding domain of the ATPase on the top surface of the protein. Those mAbs that were found to inhibit steady-state ATPase activity were found to bind to epitopes in the nucleotide binding domain of the ATPase.     

Comparative sequence and immunochemical analyses of murine monoclonal anti-morphine antibodies

A more complete characterization is given for four previously reported anti-morphine monoclonal antibodies that bind the hapten with high affinity and to which anti-idiotypic antibodies have been raised that mimic opiates at receptor binding sites. The variable (V) region nucleotide sequences of the heavy (H) and light (L) chains of these murine antibodies have been determined by direct sequencing of the poly(A)+ mRNAs using specific oligonucleotide primers and dideoxynucleotide chain-termination, and the deduced amino acid sequences are compared. The primary sequences predicted for the VH segments of 10C3 and 11C7 antibodies are closely associated with the VHIIIB subgroup of mouse H-chain (80 to 82% homology), while those for the V-regions of 3B9 and 12D4 H-chains correlate well with the VHIIC subgroup (64 to 67% homology). The 11C7, 10C3, 3B9 and 12D4 hybridoma cell lines use JH1, JH2, JH3 and JH4 DNA segments, respectively. Since considerable variations in length and primary sequence in the CDR3 (complementarity determining region) peptides of all the H-chains are evident, conservation of the D-region structure does not appear to be necessary for effective hapten binding. However, sequence homologies of the CDR2 regions of all the antibodies indicate that residues Glu H-50, Ile H-51, Pro H-52a and Tyr H-59 are conserved, and that these segments may be more critically involved in binding than the other H and L-chain hypervariable regions. The marked VL sequence homology, greater than 93%, among the L-chains and consensus lambda sequence, suggests derivation from a similar or identical VL germ-line gene. The L-chain J-region peptides for all the antibodies are classified JL1 and no VL-JL junctional diversity was apparent. The antimorphine antibody L-chains are apparently generated by the joining of a specific J-gene segment to a single germ-line V-gene segment, and minor sequence variations are the result of somatic mutations within the coding region. The leader sequence for one of the H-chains was determined. The inhibition of morphine binding by phenoxybenzylation or iodination of the affinity-purified immunoglobulins indicates the involvement of a single tyrosyl residue within or close to the antibody-combining site for the opiate. This conclusion is supported by the sequence data and nuclear magnetic resonance measurements reported in the accompanying paper, in which the results are used to interpret nuclear magnetic resonance measurements on one of the ligand-antibody systems. The possible importance of additional contact amino acids, tryptophan, aspartic and/or glutamic acids, is discussed.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.     

Acidic residues critical for the activity and biological function of yeast DNA polymerase eta

Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases. The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent. We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function. Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo. The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting. To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species. Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation. Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities. From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.     

The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins.

Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68).     

Studies on the Topography of the Catalytic Site of Acetylcholinesterase Using Polyclonal and Monoclonal Antibodies

Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.     

Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin.

Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis. Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins. Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA. The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers. Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way. Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence). This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization.     

The complete primary structure of HLA-Bw58

Serological studies indicate that HLA-B17 molecules are unusually cross-reactive with products of the HLA-A locus. In particular, a mouse monoclonal antibody MA2.1 defines an epitope that is shared by HLA-A2 and the two subtypes (Bw57 and Bw58) of B17. To investigate these relationships at the structural level, we have isolated a gene coding for Bw58 from the WT49 B cell line. The gene was transfected into mouse L cells and its protein product was characterized with a panel of monoclonal anti-HLA antibodies. The nucleotide sequence of 3520 base pairs of DNA encompassing the seven exons coding for Bw58 and associated introns was determined. The deduced protein sequences for Bw58 and eight other HLA-A,B,C molecules were compared. In the first polymorphic domain (alpha 1), Bw58 is unusual in that it is as homologous to HLA-A locus products as to HLA-B locus products. In the second polymorphic domain (alpha 2), Bw58 has greater homology to B locus products. In the alpha 1 domain of Bw58, small segments of amino acid and nucleotide sequence homology with A2 (residues 62-65) and with Aw24 (residues 75-83) are found in the major region of polymorphic diversity (residues 62-83). These similarities provide structural correlates for the serological relationships between Bw58 and A locus molecules, with residues 62-65 possibly being involved in the MA2.1 epitope. From comparisons of four HLA-A and four HLA-B sequences, there is a difference in the patterns of variation for A and B locus molecules. For B locus molecules there is greater variation in the alpha 1 domain than in the alpha 2 domain. For A locus molecules, variation in the two domains is similar and like that for B locus alpha 2 domains. In comparison to other HLA-A,B,C genes, novel inverted repeat sequences were found in the nucleotide sequence of HLA-Bw58. These sequences flank the putative RNA splicing sites at the 3' end of the exons encoding the alpha 2 and alpha 3 protein domains.     

Primary structure of the potato virus X genome: the region preceding the capsid protein cistron

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.     

Nucleotide sequence encoding the 5'' end of Xenopus laevis 18S rRNA.

We have sequenced a region of cloned Xenopus laevis ribosomal DNA encompassing the last 24 nucleotides of the external transcribed spacer and the first 275 nucleotides of the 18S gene. The start of the 18S gene was identified by correlating the results obtained from RNA hybridization and fingerprinting with the DNA sequence. This 5' region of 18S rRNA contains five 2'-O-methyl groups and at least six pseudouridine residues. Several of these modified nucleotides are clustered into a relatively short region from nucleotides 99-124. Nucleotides 227-250 constitute a distinctive sequence of 24 consecutive G and C residues. Comparison with the first 160 nucleotides of a yeast 18S gene (25) reveals three blocks of high sequence homology separated by two short tracts where homology is low or absent. The external transcribed spacer sequences diverge widely from within a few nucleotides of the start of the 18S gene.