Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 37 records · Page 2Linked to original sources

PubMed2001

Analysis of early promoters of the Bacillus bacteriophage GA-1.

Bacteriophage GA-1, which infects Bacillus sp. strain G1R, is evolutionarily related to phage phi29, which infects Bacillus subtilis. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of phi29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.

Bacillus Phages↗

PubMed2001

Phi29 family of phages.

Continuous research spanning more than three decades has made the Bacillus bacteriophage phi29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to phi29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5' ends. These phi29-like phages can be divided into three groups. The first group includes, in addition to phi29, phages PZA, phi15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of phi29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of phi29-like phages, which is included in this review.

Amino Acid Sequence↗

PubMed2001

[Seroepidemiology of Herpes simplex virus type 2 in the Amerindian Yukpa population of Zulia state, Venezuela].

BACKGROUND: Yukpa aborigines start early in life their sexual activity and promiscuity with native and foreign individuals is common among women. This behavior allows the installation and propagation of Herpes simplex virus type 2 (HSV-2). AIM: To determine the presence of antibodies to HSV-2 in a Yukpa Amerindian community at Zulia State, Venezuela. SUBJECTS AND METHODS: We studied 96 natives, between 13 and 49 years old (62.3% of the whole population, 100% of individuals in fertile age). Control population consisted of 76 individuals, with similar sex and age range distribution, of the urban population from Maracaibo, Zulia State, Venezuela. HSV-2 serologic status was determined by an indirect fluorescent antibody test. RESULTS: Antibodies to HSV-2 were detected in 53.1% of Yukpa population and 21.1% of urban population. Aboriginal women aged 21 to 40 years old had a prevalence of antibodies against HSV-2 of 66.7%. Among pregnant native women, the prevalence of antibodies against HSV-2 was 54.6%. CONCLUSIONS: There is a high prevalence of antibodies against HSV-2 among Yukpa natives in Venezuela.

Adolescent↗

PubMed2000

Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication.

The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer. The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP. Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA. We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments. Efficient initiation only requires the terminal repetition 5'-AA. The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position. Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.

Bacillus Phages↗

PubMed2000

The putative coiled coil domain of the phi 29 terminal protein is a major determinant involved in recognition of the origin of replication.

The linear double-stranded genome of phage phi29 contains a terminal protein (TP) covalently linked at each 5' DNA end, called parental TP. Initiation of phi29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing phi29 DNA polymerase and primer TP. It has been argued that origin recognition involves protein-protein interactions between parental and primer TP. Analysis of the TP sequence revealed that the region between amino acids 84 and 118 has a high probability to form an amphipatic alpha-helix that could be involved in the interaction between parental and primer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted amphipatic alpha-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic alpha-helix of TP is an important determinant involved in origin recognition.

Amino Acid Sequence↗

PubMed2000

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

Amino Acid Motifs↗

PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗

PubMed2000

Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1.

The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.

Adenosine Monophosphate↗

PubMed2000

Identification of residues within two regions involved in self-association of viral histone-like protein p6 from phage theta29.

Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.

Amino Acid Sequence↗

PubMed2000

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7.

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Bacillus Phages↗

PubMed2000

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins.

DNA replication of phi29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of phi29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of phi29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during phi29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by phi29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the phi29 and Nf SSBs do not show any effect. On the other hand, the phi29 SSB is the only one of the three SSBs able to increase the replication rate of phi29 DNA polymerase in primed M13 ssDNA replication. From the fact that the phi29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of phi29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on phi29-like DNA replication has been proposed.

Amino Acid Sequence↗

PubMed2000

Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication.

The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer. The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends. The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement. The same mechanism is used by the related phage Nf. However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA. To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage. Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.

Bacteriophages↗

PubMed2000

Structural and functional comparative study of the complexes formed by viral ø29, Nf and GA-1 SSB proteins with DNA.

Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, ø29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ø29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ø29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, K(eff)=18.6 x 10(5) M(-1); o29 SSB, K(eff)=2.2 x 10(5) M(-1); Nf SSB, K(eff)=2.9 x 10(5) M(-1)). Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ø29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ø29 DNA amplification.

Bacillus↗

PubMed2000

Analysis of O29 DNA polymerase by partial proteolysis: binding of terminal protein in the double-stranded DNA channel.

ø29 DNA polymerase, which belongs to the family of the eukaryotic type DNA polymerases, is able to use two kinds of primers to initiate DNA replication: DNA and terminal protein (TP). By partial proteolysis we have studied the regions of ø29 DNA polymerase involved in primer binding. With proteinase K, no change in the proteolytic pattern was observed upon DNA binding, suggesting that it does not induce a global conformational change in ø29 DNA polymerase. Conversely, two of the three main cleavage sites obtained by partial digestion of free ø29 DNA polymerase with endoproteinase LysC were protected upon DNA binding, indicating that the DNA could be occluding these cleavage sites to the protease either directly by itself and/or indirectly by induction of local conformational changes affecting their exposure. Partial proteolysis with endoproteinase LysC of ø29 DNA polymerase/TP heterodimer resulted in a protection and digestion pattern similar to that obtained with DNA, suggesting that both primers, DNA and TP, fit in the same double-stranded DNA-binding channel and protect the same regions of ø29 DNA polymerase.

Bacillus Phages↗

PubMed2000

Pleiotropic effect of protein P6 on the viral cycle of bacteriophage phi29.

The product of bacteriophage phi29 early gene 6, protein p6, is a double-stranded-DNA binding protein and one of the more abundant proteins during viral infection. We have studied the role of protein p6 in vivo through the infection of suppressor and nonsuppressor Bacillus subtilis strains with a phage carrying a nonsense mutation in gene 6, sus6(626). In the absence of functional protein p6, the two major processes of the viral cycle, transcription and DNA replication, were affected. Viral DNA synthesis was practically abolished, and early transcription was remarkably delayed and, in addition, underregulated at late times of the infection. The amount of protein p6 synthesized after infection with mutant phage sus6(626) under suppressor conditions was sixfold lower than that produced after wild-type infection. Nonetheless, phage production was as high as that obtained after wild-type infection. These results indicate that p6 is synthesized in amounts higher than those needed for most of its functions. However, the concentration of protein p6 appeared to be important for repression of the early promoter C2.

Bacillus Phages↗

PubMed2000

Quantitative and functional study of breast cancer axillary lymph nodes and those draining other human malignant tumors.

Lymphocyte functional activity from lymph nodes draining human malignancies reflects the host immune response against tumour. Breast cancer is the neoplasia with the greatest amount of identified antigens but a weak inducer of a host efficient immune response. In our study we compared the mitogen stimulated-proliferative response of cells isolated from metastases-free lymph nodes draining breast cancer (Group 1), other malignant tumours (Group 2), and those obtained from patients without malignancies (Control group). A significant decrease of the proliferative response in cells isolated from lymph nodes draining breast cancer was observed comparing it to the other groups. Quantitative analysis of B and T cells showed a higher number of B cells than T cells in Groups 1 and 2. Moreover, Group 1 presented a two fold increase of T cells compared with Group 2. Our results suggest that the immunosuppression observed in lymph nodes draining breast cancer is higher than the inmunosuppression presented in other malignant tumours and that impaired function is not correlated with the increased number of T cells.

Adult↗

PubMed1999

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Arginase↗

PubMed1999

Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.

The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head-on. However, head-on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery. Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase.

Bacillus Phages↗