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J C Alonso

Publications and source records attributed to J C Alonso.

At least 19 recordsLinked to original sources
PubMed1999

A2 cro, the lysogenic cycle repressor, specifically binds to the genetic switch region of Lactobacillus casei bacteriophage A2.

Lysogenic induction of temperate bacteriophage A2 of Lactobacillus casei is controlled by the action of its cI and cro products at the phage operator region. Three 20-bp inverted repeated DNA segments (subsites O1, O2, and O3) and the two divergent (PL and PR) promoters were mapped within the 153-bp operator region. The A2-encoded Cro product is shown to be the functional homolog of lambda Cro. The binding of Cro to the three operator subsites is noncooperative and yields two discrete protein-DNA complexes of retarded migration in mobility shift assays. The Kapp value for the Cro-PL-PR DNA complex was estimated to be 6 nM. Cro shows a slightly higher affinity for O3 than for O1 and O2 subsites. The O3 subsite overlaps the -35 hexamer of the PL promoter, which directs cI expression. A Cro mutant protein, devoid of the last 12 residues (Cro*), allowed the assignment of the DNA-binding domain to the NH2 end of Cro. The C end enhances its affinity for the DNA and probably stabilizes bending induced by Cro.

Base Sequence↗
PubMed1999

Bacillus subtilis sequence-independent DNA-binding and DNA-bending protein Hbsu negatively controls its own synthesis.

Transcription of the hbs gene under vegetative growth condition is subject to repression when cells enter in late exponential phase. We have determined the sites at which transcription of the hbs gene initiates in vitro. On a supercoiled template, transcription of the hbs gene is initiated by sigmaARNAP at two overlapping hbs promoters (P1 and P3). We have demonstrated that highly purified Hbsu protein acts as a repressor of its own synthesis. The binding of the sequence-independent DNA-binding and DNA-bending Hbsu protein does not seem to exclude sigmaARNAP from the promoters. In this report we show that Hbsu, in vitro, does not repress transcription by a mere steric hindrance on sigmaARNAP binding.

Artificial Gene Fusion↗
PubMed1999

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Adenosine Triphosphate↗
PubMed1999

The prokaryotic beta-recombinase catalyzes site-specific recombination in mammalian cells.

The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.

Animals↗
PubMed1999

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function.

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec+ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups alpha (recF, recR and recL strains), beta (addA5 addB72), gamma (recH342) and epsilon (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group alpha and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex.

Bacillus subtilis↗
PubMed1999

Proteolytic cleavage of gram-positive beta recombinase is required for crystallization.

Beta recombinase, a DNA resolvase-invertase, catalyzes in the presence of a chromatin-associated protein such as Hbsu, DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented target (six) sites. Single crystals of the beta recombinase from plasmid pSM19035 were obtained using the vapor diffusion technique with ammonium phosphate as the precipitating agent. The crystals diffracted X-rays to a maximum resolution of 2.5A. Due to proteolytic degradation during the crystallization experiment, the crystals contain only the N-terminal catalytic domain of beta recombinase corresponding to about 60% of the molecular mass of the initially assayed native protein. The proteolytic removal of the C-terminal DNA-binding domain demonstrated that protein modification can be essential to provide material suitable for X-ray analysis.

Binding Sites↗
PubMed1999

Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2.

The temperate bacteriophage A2 forms stable lysogens in Lactobacillus casei. The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR. These promoters mediate transcription from cI and a putative cro, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment. CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the -35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR. CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O1 and O2, enhances the positioning of the RNA polymerase with the PL promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage.

Bacteriophages↗
PubMed1999

Technetium-99m-HMPAO-labeled leukocyte imaging in patients with seronegative spondyloarthropathies.

OBJECTIVE: Gut inflammation is frequent among patients with seronegative spondyloarthropathies (SSp). The purpose of this study was to evaluate the presence of positive abdominal findings in patients with SSp who did not have clinical symptoms or signs of inflammatory bowel disease (IBD). This represents a new indication for abdominal 99mTc-HMPAO-labeled leukocyte scintigraphy. METHODS: Eighty-six patients (59 with SSp and 27 controls) were prospectively imaged with 99mTc-HMPAO-labeled leukocytes. RESULTS: Leukocyte imaging was positive in 33 patients with SSp (56%), 27 of these patients scored between 2+ and 4+ (51%). Four (15%) control patients also had positive findings. CONCLUSION: These findings provide evidence linking SSp with intestinal inflammation. SSp may be an important new indication for 99mTc-HMPAO-labeled autologous leukocyte scintigraphy.

Adult↗
PubMed1998

Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase-ssDNA complex.

Purified Bacillus subtilis DnaG primase (predicted molecular mass 68.8 kDa) behaves as a monomer in solution. We demonstrate that DnaG physically interacts with bacteriophage SPP1 hexameric helicase G40P (G40P6) in the absence of ATP. G40P6-ATP forms an unstable complex with ssDNA, and by itself carries out ATP-driven translocation along a ssDNA template with low processivity. The presence of DnaG in the reaction mixture increased the helicase activity of G40P6 about 3-fold, but not the ATPase activity. The results presented here suggest that the DnaG protein stabilises the G40P6-ssDNA complexes.

Adenosine Triphosphatases↗
PubMed1998

Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P).

The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication. The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate. We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein. Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views. Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture. These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole. A clear structural handedness has also been detected in some of these forms. An analysis of these quaternary states and a model for the structural organization of G40P are presented.

Bacillus subtilis↗
PubMed1998

beta Recombinase catalyzes inversion and resolution between two inversely oriented six sites on a supercoiled DNA substrate and only inversion on relaxed or linear substrates.

The beta recombinase, in the presence of a chromatin-associated protein such as Hbsu, catalyzes DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented six sites. Hbsu stabilizes the formation of the recombination complex (Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945). In this study we show that resolution by beta recombinase strictly requires supercoiled DNA, but inversion does not. On a substrate with two inversely oriented six sites, beta recombinase catalyzed both resolution and inversion if the DNA was supercoiled but only inversion if the substrate was relaxed or linear. Hbsu was critical for the formation of synaptic complexes; its concentration relative to that of the supercoiled DNA substrate determined whether resolution or inversion products were preferentially formed. The results suggest that the beta recombinase forms unproductive short-lived synaptic complexes between two juxtaposed inversely oriented six sites; the presence of 3 to 13 Hbsu dimers per supercoiled DNA molecule would stabilize a synaptic complex with a relative geometry of the six sites allowing beta recombinase preferentially to achieve resolution. Supercoiling probably helps to overcome an energetic barrier, since resolution does not occur in relaxed DNA. The presence of >30 Hbsu dimers per DNA molecule probably favors the formation of a recombination complex with a different geometry since the reaction is directed preferentially toward DNA inversion.

Catalysis↗
PubMed1998

Characterization of the small subunit of the terminase enzyme of the Bacillus subtilis bacteriophage SPP1.

The small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix-turn-helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene 1 of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure.

Amino Acid Sequence↗
PubMed1998

Basic and acidic regions flanking the HMG domain of maize HMGa modulate the interactions with DNA and the self-association of the protein.

The maize HMGa protein is a typical member of the family of plant chromosomal HMG1-like proteins. The HMG domain of HMGa is flanked by a basic N-terminal domain characteristic for plant HMG1-like proteins, and is linked to the acidic C-terminal domain by a short basic region. Various derivatives of the HMGa protein were expressed in Escherichia coli and purified. The individual HMG domain can functionally complement the defect of the HU-like chromatin-associated Hbsu protein in Bacillus subtilis. The basic N-terminal domain which contacts DNA enhances the affinity of the protein for linear DNA, whereas it has little effect on the structure-specific binding to DNA minicircles. The acidic C-terminal domain reduces the affinity of HMGa for linear DNA, but does not affect to the same extent the recognition of DNA structure which is an intrinsic property of the HMG domain. The efficiency of the HMGa constructs to facilitate circularization of short DNA fragments in the presence of DNA ligase is like the binding to linear DNA altered by the basic and acidic domains flanking the HMG domain, while the supercoiling activity of HMGa is only slightly influenced by the same regions. Both the basic N-terminal and the acidic C-terminal domains contribute directly to the self-association of HMGa in the presence of DNA. Collectively, these findings suggest that the intrinsic properties of the HMG domain can be modulated within the HMGa protein by the basic and acidic domains.

Animals↗
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↗