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J C Alonso

Publications and source records attributed to J C Alonso.

At least 55 records · Page 3Linked to original sources
PubMed1998

Four differently chromatin-associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes.

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific beta-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their 'extractability' from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.

Chromatin↗
PubMed1998

Genetic recombination in Bacillus subtilis 168: effects of recU and recS mutations on DNA repair and homologous recombination.

Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed "recU" and "recU1" (recU:cat) and "recS" and "recS1" (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the alpha (recF), beta (addA5 addB72), gamma (recH342), and epsilon (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (alpha epistatic group), addAB (beta), recH (gamma), recU (epsilon), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed "zeta".

Bacillus subtilis↗
PubMed1997

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the 'heavy strand' is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have shown that 25 orfs are essential. Of the remaining orfs, functions could be predicted for the products of five of the orfs on the basis of comparison of the deduced amino acid sequence to known proteins. Intergenic regions include most of the 5 PE and the 4 PL promoters. Transcripts are polycistronic. Transcription from the PE promoters is mediated by host RP, whereas recognition of the PL promoters requires an additional unidentified phage-encoded product. Translation of mRNA transcribed from most of the orfs seems to be initiated independently, each from its own ribosomal binding and initiation site, although a few cases of coupled translation have been reported. The organization of SPP1 genes involved in the replication, DNA packaging and phage assembly proteins resembles the organization of genes of equivalent regions of different E. coli double-stranded DNA phages. Absence of aa sequence similarity between analogous proteins of different phages suggested that the conserved gene organization is representative of a primordial bacteriophage.

Bacillus Phages↗
PubMed1997

The recombinant product of the Chryptomonas phi plastid gene hlpA is an architectural HU-like protein that promotes the assembly of complex nucleoprotein structures.

The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.

Bacillus subtilis↗
PubMed1997

Purification and characterization of the RecF protein from Bacillus subtilis 168.

Genetic evidence suggests that the Bacillus subtilis recF gene product is involved in DNA repair and recombination. The RecF protein was overproduced and purified. NH2-terminal protein sequence analysis of RecF was consistent with the deduced amino acid sequence of the recF gene. The RecF protein (predicted molecular mass 42.3 kDa) bound single- and double-stranded DNA in a filter binding and in a gel retarding assay. The RecF-ssDNA or -dsDNA complex formation proceeds in the absence of nucleotide cofactors. RecF-ssDNA interaction is markedly stimulated by divalent cations. The apparent equilibrium constants of the RecF-DNA complexes are approximately 110-130 nM for both ssDNA and dsDNA. The binding reaction shows no cooperativity. The RecF protein does not physically interact with the RecR protein. Under our experimental conditions an ATPase activity was not associated with the purified RecF protein or with the RecF and RecR proteins.

Adenosine Triphosphatases↗
PubMed1997

The replisome organizer (G38P) of Bacillus subtilis bacteriophage SPP1 forms specialized nucleoprotein complexes with two discrete distant regions of the SPP1 genome.

Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P). G38P specifically binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome. One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin (oriL). The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR. Both sites contain two types of repeated elements (termed Box AB and A + T-rich region). The K(app) for the G38P-oriL DNA and G38P-oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively. G38P binds to the distant oriL and oriR sites cooperatively. DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A + T-rich region. Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through protein-protein interaction. G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it. These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted.

Bacillus Phages↗
PubMed1997

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted. We have assigned genes 7, 8, 9, 11, 12, 13, 14 and 15 to these orfs by correlating genetic and immunological data with DNA and protein sequence information. G7P was identified as a minor structural component of proheads and heads, G11P as the scaffold protein, G12P and G15P as head minor proteins and G13P as the coat protein. Characterization of intermediates in head assembly, which accumulate during infection with mutants deficient in DNA packaging or in morphogenetic genes, allowed the definition of the head assembly pathway. No proteolytic processing of any of the head components was detected. Removal of G11P by mutation leads to the accumulation of prohead-related structures and aberrant particles which are similar to the assemblies formed by purified G13P in the absence of other phage-encoded proteins. The native molecular masses of G11P and G13P are about 350 kDa and larger than 5000 kDa, respectively (predicted molecular masses 23.4 kDa and 35.3 kDa, respectively). G13P, upon denaturation and renaturation, assembles from protomers into some prohead-related structures. The organization of the DNA packaging and head genes of SPP1 resembles the organization of genes in the analogous regions of phage lambda and P22.

Amino Acid Sequence↗
PubMed1997

Bacillus subtilis 168 RecR protein-DNA complexes visualized as looped structures.

The Bacillus subtilis 168 RecR protein bound to duplex DNA in the presence of ATP and divalent cations (Mg2+ and Zn2+) was visualized by electron microscopy as a nearly spherical particle. A RecR homomultimer is frequently located at the intersection of two duplex DNA strands in an interwound DNA molecule, generating DNA loops of variable length. Two individual DNA molecules bound to the same protein are seen at a very low frequency, if at all. The association of RecR with the intersection of two duplex DNA strands is more often seen in supercoiled than with relaxed or linear DNA. The RecR protein displays a slight but significant preference for negatively supercoiled over linear DNA. The minimum substrate size for RecR protein is about 150 bp in length. A possible mechanism for RecR function in DNA repair is discussed.

Bacillus subtilis↗
PubMed1997

[Evaluation of activities and effectiveness of an asthmatic child program developed in primary care].

OBJECTIVE: To evaluate activities and effectiveness of a programme of attention to asthmatic child, defined by a proper diagnostic process and therapeutic intervention, by the gain of educational goals and by reduction of number of asthma attacks. DESIGN: A before-after study SETTING: Primary care PARTICIPANTS: The first 81 children attracted to the programme. Aged 5 to 13. INTERVENTION: Each child was periodically revised (mean time of monitoring 11 months and 24 days: interval 7-17 months). MEASUREMENTS AND MAIN RESULTS: Intervention obtained significative results (p < 0.0001) in: lung function study and allergens detection, use of inhalatory way and peak flow meter. Asthma attacks were reduced (p < 0.0001): before 216, mean 2.66, SD 1.85, range 0-8; and after 117, mean 1.44, SD 1.27, range 0-5. CONCLUSIONS: 1. A systematic and organized intervention from Primary Care results effective to improve quality of life in asthmatic child (better diagnostic, more abilities, less asthma attacks). 2. It must be stated new strategies for reduction of smoking at home of these children.

Adolescent↗
PubMed1997

Bacillus subtilis bacteriophage SPP1 terminase has a dual activity: it is required for the packaging initiation and represses its own synthesis.

The B. subtilis bacteriophage SPP1 terminase, encoded by genes 1 and 2, is required for the initiation of headful packaging. The DNA segment to which gene 1 product (G/P) binds includes the pacL and pacR sites and the late PL1 and PL2 promoters from which genes 1 to 7 are transcribed. When SPP1wt or SPP1sus115 (gene 6-) phages were used to infect a B. subtilis sup0 strain, the gene 1 to 7 mRNA synthesis was reduced at late times of infection. This was not observed, however, when either chloramphenicol was added 7 min after infection with SPP1wt or when SPP1sus114 (gene 1-) or SPP1sus19 (gene 2-) were used to infect B. subtilis sup0 cells. These results suggest that the terminase enzyme functions as a repressor of its own transcription. G/P and B. subtilis RNA polymerase (RP) bind to the pacL segment, which contains the PL1 and PL2 promoter region. The binding of G/P to the pacL site does not seem to exclude RP from the promoters, despite of the overlapping of their binding sites. It is likely that the terminase protein does not repress transcription by a mere steric hindrance of RP binding.

Bacillus Phages↗
PubMed1997

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the beta recombinase of pSM19035.

The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.

Amino Acid Sequence↗
PubMed1997

Characterization of an lrp-like (lrpC) gene from Bacillus subtilis.

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilis lrp-like-gene is a bona fide Lrp protein-the first one to be detected in gram-positive bacteria. When expressed in E. coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B subtilis Lrp-like protein plays a role in the growth phase transition.

Amino Acid Sequence↗
PubMed1997

RecR is a zinc metalloprotein from Bacillus subtilis 168.

The Bacillus subtilis RecR protein is required for DNA repair and recombination in vivo. In its N-terminal portion, RecR possesses potential zinc-ligand structures associated with the multicysteine (C4) superfamily. The number and arrangement of the cysteine residues is suggestive of RecR being a zinc-finger protein. One of the four cysteines (Cys-60) has been replaced by a Ser (C60S) or an Ala (C60A) residue to generate the recR60 and recR601 genes, respectively. B. subtilis recR60, recR601 or delta recR1 (a null-mutant allele) cells are 10-, 134- and 144-fold more sensitive to 10 mM methanesulphonate and 95-, 900- and 1100-fold more sensitive to the lethal effect of 100 microM 4-nitroquinoline-1-oxide (4NQO) than the wild-type strain, respectively. The RecR zinc-ligand C4 motif does not seem to be accessible, because the protein is highly resistant to oxidation and moderately resistant to reduction. We have determined by different biochemical methods that RecR is a zinc metalloprotein whose cysteine residues have a structural and/or functional role.

Amino Acid Sequence↗
PubMed1997

The Bacillus subtilis chromatin-associated protein Hbsu is involved in DNA repair and recombination.

The Bacillus subtilis hbs gene encodes an essential chromatin-associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non-specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site-directed mutants in the hbs gene and used these mutant genes to substitute the wild-type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe-47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg-55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and beta protein-mediated site-specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recH and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA-damaging agents methylmethane sulphonate (MMS) or 4-nitroquinoline-1-oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20- to 50-fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region.

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.

Amino Acid Sequence↗
PubMed1996

The ColE1 unidirectional origin acts as a polar replication fork pausing site.

Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.

Bacteriocin Plasmids↗
PubMed1996

Site-specific recombination in gram-positive theta-replicating plasmids.

This review summarises current information on the site-specific recombinases encoded by the plasmids of the Gram-positive bacteria that have low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of the recombination systems encoded by the theta-replicating plasmids and compares them with the site-specific recombinases encoded by transposons or plasmids originally isolated from Gram-negative bacteria.

Amino Acid Sequence↗