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J C Alonso

Publications and source records attributed to J C Alonso.

At least 73 records · Page 4Linked to original sources
PubMed1996

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.

Bacterial Proteins↗
PubMed1996

The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination.

Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair. When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced. B. subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins. In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E. coli MFD protein. Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively. The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa). The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity. The Mfd protein was able to displace in vitro B. subtilis or E. coli RNA polymerase stalled at a lesion. Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA. In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.

Amino Acid Sequence↗
PubMed1996

Abdominal scintigraphy using 99mTc-HMPAO-labelled leucocytes in patients with seronegative spondylarthropathies without clinical evidence of inflammatory bowel disease.

Abdominal scintigraphy with technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO)-labelled leucocytes is an excellent tool for evaluating disease extent and activity of intestinal lesions in patients with inflammatory bowel disease (IBD). In some cases of seronegative spondylarthropathies (SSp), IBD may remain subclinical. The aim of this study was to evaluate the presence of positive abdominal scintigraphy in patients with SSp and without clinical symptoms or signs of IBD. To this end we studied 32 patients with active SSp (European Spondylarthropathy Study Group 1991 criteria) without clinical evidence of IBD (eight had ankylosing spondylitis, four psoriatic arthritis, three reactive arthritis an 17 undifferentiated SSp) and 11 controls without SSp. All SSp and control patients received similar doses of non-steroidal anti-inflammatory drugs (NSAIDs). Abdominal scintigraphic images were obtained at 30 and 120 min after re-injection of 99mTc-HMPAO-labelled leucocytes. The 99mTc-HMPAO-labelled leucocyte scan was positive in 17 patients with SSp (53.1%) (six with ankylosing spondylitis, three with psoriatic arthritis, two with reactive arthritis and six with undifferentiated SSp). Fourteen patients scored from 2 to 4 on the intensity of uptake scale. The colon and terminal ileum were predominantly involved. Axial involvement was more frequent in patients with a positive scan than in patients with negative results (P<0.05) (64.7% vs 26.6%; odds ratio: 5). No control patient showed a positive scan. It is concluded that 99mTc-HMPAO-labelled leucocyte scan shows increased uptake among patients with SSp without evidence of IBD. These findings provide new evidence linking SSp with intestinal inflammation and suggest that in some cases a bowel-related process could contribute to the development of SSp. Long-term follow-up studies with more patients are necessary to evaluate the diagnostic and therapeutic implications of these results.

Abdomen↗
PubMed1996

Broad-host-range plasmid replication: an open question.

Many factors can influence the ability of plasmids to colonize different hosts, efficient replication probably being the most critical one. Two major strategies seem to facilitate promiscuous plasmid replication: (i) initiation independent of host initiation factors; and (ii) versatile communication between plasmid and host initiation factors. Appropriate communication between a replicon and the different hosts, which becomes crucial at the initation of plasmid replication, plays a major role in plasmid promiscuity. Fused replicons or mechanisms that rescue collapsed replication forks may increase the efficiency of plasmid propagation. However, their contribution to plasmid promiscuous replication remains to be fully evaluated. Several examples of host-specific adaptation of promiscuous plasmids point to an enormous flexibility of these replicons.

DNA Replication↗
PubMed1996

[Gammagraphy with HMPAO-99mTc leukocytes in intestinal inflammatory disease].

The value of 99mTc-hexamethylpropyleneamine oxime (HMPAO) leukocyte images has been confirmed in patients with Inflammatory Bowel Disease. We performed 121 scintigraphic examination. The results were evaluated by radiologic and/or endoscopic examinations. The patients were divided into three subgroups: A) patients with active disease and less than 15 days of corticotherapy (n = 42). B) patients with active disease and more than 15 days of corticotherapy (n = 31). C) asymptomatic patients (n = 48). Over all sensitivity and specificity were 86% and 92.2% respectively. The sensitivity in subgroup A was 97.5% and 70% in the subgroup B. In the subgroup A the isotopic study showed the entire area of active inflammation in 95% (38/40) and colonoscopy in 32.5% (13/40). The leucocyte scanning showed clear pathologic activity in the ileum with a sensitivity of 86%. We conclude that 99mTc-leukocyte scintigraphy is the best method to assess the location of active inflammation in patients with less than 15 days of treatment. Scintigraphy was best in the assessment of active disease especially in Crohn's disease of the small bowel.

Adult↗
PubMed1995

The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region.

Initiation of SPP1 DNA packaging requires the gene 1 and gene 2 products (G1P and G2P), which are different subunits of the terminase enzyme. G1P specifically recognizes the phage packaging initiation region (pac). The apparent equilibrium constant for the G1P-pac-DNA complex was estimated to be 9 nM. DNase I footprinting experiments reveal that the pac region can be subdivided into three discrete sites (pacL, pacC and pacR). G1P binds co-operatively to the non-adjacent pacL and pacR sites. Several G1P protomers bind to the target sequences which map close to the pac cleavage site (pacC site), but do not overlap with it. G1P interacts in a different fashion with the encapsidated (pacR site) and with the non-encapsidated (pacL site) end of the phage genome. G1P interaction with the intrinsically bent pacL DNA occurs only on one face of the DNA double helix. G1P binding to the pacL and in the pacR region results in a DNA loop. Electron microscopy of purified G1P shows that the protein is an oligomer in solution. G1P binding to the core region of the pacL site could facilitate the formation of a higher-order nucleoprotein structure. This specialized complex would allow the pac DNA to form a loop between binding sites brought together by interaction with G1P. The results presented here suggest that G1P could provide a tool to discriminate the first encapsidated end, which contains pacR, from the non-encapsidated pacL end.

Bacillus Phages↗
PubMed1995

The beta recombinase of plasmid pSM19035 binds to two adjacent sites, making different contacts at each of them.

The beta recombinase from plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented six sites in the presence of a chromatin-associated protein (Hbsu, HU or HMG-1). The six site is a DNA segment containing two binding sites (I and II) for beta protein dimers. We show that beta recombinase binds sequentially to both sites, having a different affinity for each one. Hydroxyl radical footprints show a different protection pattern at each site. Positions critical for beta protein binding have been identified by methylation interference and missing nucleoside assays. The results indicate that the protein recognizes each site in a different way. Comparison of the beta protein recombination site with that of DNA resolvases and DNA invertases of the Tn3 family, to which it belongs, shows that these sequences can be divided into two regions. One corresponds to the crossover point and is similar for all recombinases of the family. The other region differs in the different subfamilies and seems to have an architectural role in aligning the crossover sites at the synaptic complex. The different ways to assemble this complex could explain why each system leads to a particular recombination event: DNA resolution (resolvases), inversion (invertases) or both (beta recombinase).

Bacterial Proteins↗
PubMed1995

Plasmid rolling circle replication and its control.

This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.

Bacterial Proteins↗
PubMed1995

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number.

Plasmids control their copy number by limiting the amount of the initiator for DNA replication. The plasmid pUB110 initiator protein is termed RepU. Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene. The RepU/RepU* protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants.

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (alpha to theta) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.

Amino Acid Sequence↗
PubMed1995

The Bacillus subtilis histone-like protein Hbsu is required for DNA resolution and DNA inversion mediated by the beta recombinase of plasmid pSM19035.

The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been identified as the Bacillus subtilis Hbsu protein. Hbsu is a nonspecific DNA-binding and DNA-bending protein. The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule. DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated. HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable. In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E. coli cells, but failed to do so in an HU-deficient strain. The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.

Bacillus subtilis↗
PubMed1995

Different abdominal scintigraphy pattern in patients with ulcerative colitis, Crohn's disease and seronegative spondylarthropathies.

The aim was to analyse the abdominal scintigraphy pattern in patients with seronegative spondylarthropathy (SSp), ulcerative colitis (UC) and Crohn's disease (CD). A total of 117 patients with defined histological lesions of inflammatory bowel disease (IBD) (68 UC and 49 CD), 32 patients with active SSp [European Spondylarthropathy Study Group (ESSG) 1991 criteria] without clinical evidence of IBD and 21 controls without IBD or SSp were studied. All patients with SSp and controls received similar doses of non-steroidal anti-inflammatory drugs. Abdominal scintigraphy images were obtained at 30 and 120 min after injection of 99m-technetium hexamethyl propylene amine oxime (99mTc-HMPAO)-labelled leucocytes. The 99mTc-HMPAO-labelled leucocyte scan was positive in 17 patients with SSp (53.1%), 45 patients with UC (66.1%) and 33 patients with CD (67.3%). Rectum and sigma involvement was more frequent in patients with UC (68.8%) than in patients with SSp (23.5%) or CD (33.3%) (P < 0.05) [odds ratios (OR): 7.1 and 4.4, respectively]. Terminal ileum involvement was more frequent in patients with CD (63.6%) than in patients with SSp (23.5%) or UC (8.8%) (P < 0.05) (OR: 5.6 and 17.9, respectively). The 99mTc-HMPAO-labelled leucocyte scan shows an increased uptake in patients with SSp without evidence of IBD. Perhaps these patients represent one end of the spectrum of IBD, but rectal and terminal ileum involvement were less frequent in patients with SSp than in patients with UC or CD.

Abdomen↗
PubMed1995

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

Bacterial Proteins↗
PubMed1995

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.

Alanine↗
PubMed1994

Analysis of the Bacillus subtilis bacteriophages SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging.

Gene 1 product (G1P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and rho 15 is essential for DNA maturation and packaging. A DNA segment containing gene 1 of phage SF6 or rho 15 origin was cloned and sequenced. SF6 and rho 15 G1P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G1P of SPP1. The G1P of all three phages contains three conserved segments (I, II, and III). Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted. G1P of both SPP1 and SF6 origin was purified. SPP1 G1P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa. The 13-kDa polypeptide results from a translation start signal within gene 1, and hence was termed SPP1 G1P*. G1P of both SPP1 and SF6 binds specifically to a pac-containing DNA fragment, whereas G1P*, which lacks segment I, does not. Chimeric G1P proteins were obtained by domain swapping between gene 1 of SPP1 and SF6. The results presented here suggest that the G1P DNA binding motif lies in segment I and the major determinant for G1P::G1P interaction might lie in segment II. Segment III and the extended C-terminal part of SPP1 G1P are dispensable. The G1P::G2P interacting region remains uncharacterized.

Adenosine Triphosphatases↗
PubMed1994

Nucleotide sequence and complementation studies of the gene 35 region of the Bacillus subtilis bacteriophage SPP1.

Genetic analysis of the Bacillus subtilis bacteriophage SPP1 defective in gene 35 shows that the gene 35 product (G35P) is essential for SPP1 growth. The defect in growth of SPP1tsl17 and SPP1tsl20F at nonpermissive temperature is overcome by wild-type gene 35 expressed from a plasmid. The region where gene 35 maps was cloned and sequenced. Analysis of the nucleotide sequence (5884-bp) around gene 35 revealed 13 open reading frames (orfs). We have assigned the term gene to three of these orfs; gene 35, gene 36, the product of which shares homology with SSB proteins, and gene 38, given the gene order orf 34-orf 34.1-orf 34.2-orf34.3-orf34.4-gene 35-gene 36-orf 36.1-orf 37-orf 37.1-orf 37.2-orf 37.3-gene 38. Gene 35 encodes a protein of 32.0 kDa. By using the T7 promoter-expression system for gene 35 a radioactive band of the expected molecular mass was detected.

Bacillus Phages↗
PubMed1994

Gene organization of the Streptococcus pyogenes plasmid pDB101: sequence analysis of the orf eta-copS region.

The gene organization of the broad-host-range low-copy-number pSM19035-derived plasmid pDB101 is presented. Analysis of the 19,202-bp sequence revealed thirteen different open reading frames (orfs). Nine of these orfs (repS-orf-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1 have been previously identified [Cegłowski et al., Gene 136 (1993) 1-12]. The extraordinarily long inverted repeated sequence, which includes orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta, comprises 76% of the pDB101 molecule. The gene order in pDB101 is repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1-orf zeta-orf epsilon-orf delta-orf gamma-orf beta-orf alpha-orf eta-orf theta-orf1-copS. The organization of genes of the orf eta-orf gamma region resembles the organization of genes in the orfA-orfI region of pAM beta 1. Except for Orf1, bands of radioactive proteins corresponding to the molecular mass of the deduced reading frames (26.7, 14.3 and 10.3 kDa) were detected using the T7 promoter-expression system. The orf1 encoded a product (deduced molecular mass 28.3 kDa) which shows anomalous electrophoretical mobility corresponding to 60 kDa. The copS- and orf1-encoded proteins share homology to plasmid copy number control systems and Gram+ cocci surface proteins, respectively. The orf eta and orf theta encode proteins with unknown activity.

Amino Acid Sequence↗