Search PubMed⌕ Search

Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 253 records · Page 14Linked to original sources
PubMed1984

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.

Bacillus subtilis↗
PubMed1984

In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs.

Bacillus subtilis↗
PubMed1984

Long-term alterations in the maternal behavior of neonatally undernourished rats.

The effect of early food and sensory deprivation on the maternal responsiveness of female rats was investigated. Animals that were neonatally undernourished by daily mother-litter separation (involving both food and sensory deprivation) showed significant deficits in maternal care, consisting of a reduction in nest rating, nursing time, and retrieving responses. Moreover, they exhibited exaggerated grooming and circling movements in comparison with the controls. Dams neonatally undernourished by the nipple-ligation of their mothers (a method that minimizes sensory deprivation) displayed less alterations in maternal behavior, and no significant differences in grooming and circling from the controls. The data suggest that nest rating, nursing time, and retrieving latency are closely related to food restriction, while the frequency of grooming and circling behavior are primarily associated with sensory deprivation. These results support the view that environmental influences related to food intake and sensory stimulation, interacting at critical stages of brain development, are essential for the maturation of adult behavioral patterns.

Animals↗
PubMed1984

Nucleotide sequence at the termini of the DNA of Streptococcus pneumoniae phage Cp-1.

The 5' ends of Cp-1 DNA, which have a covalently linked terminal protein, can be partially unblocked by treatment with 1 M NaOH (E. Garcia, A. Gomez, C. Ronda, C. Escarmis, and R. Lopez (1983) Virology 128, 92-104) and labeled with polynucleotide kinase and [gamma-32P]ATP. The sequence of the first 444 and 520 nucleotides at the termini of Cp-1 DNA has been determined. A 236-nucleotide-long inverted terminal repeat was found and, in addition, the 116 nucleotides following the repeat show 93% homology. The first 352 nucleotides at both ends have an adenine plus thymine content of 75%. More than 50% of the nucleotides of the sequenced regions are involved in repeats of a minimum of 8 nucleotides. Three promoter-like sequences were also found at each end of Cp-1 DNA.

Bacteriophages↗
PubMed1984

Cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of Bacillus subtilis phage phi 29.

The phi 29 DNA restriction fragment HindIII-D, shown to contain gene 10 coding for the connector protein, has been cloned in plasmid pPLc28 under the control of the pL promoter of phage lambda. After heat induction to inactivate the lambda repressor, a protein with the electrophoretic mobility of the connector protein p10 was synthesized, accounting for about 30% of the total Escherichia coli protein after 3 h of induction. The 2205 nucleotide-long sequence of the cloned HindIII-D fragment has been determined. The sequenced region has an ORF coding for a protein of Mr 35881 that was shown to correspond to the connector protein by determination of the amino-terminal sequence of purified protein p10. Features of the nucleotide sequence and the amino acid sequence of protein p10 are discussed.

Amino Acid Sequence↗
PubMed1984

Template requirements for initiation of phage phi 29 DNA replication in vitro.

The template requirements for the formation of the phi 29 protein p3-dAMP initiation complex in vitro have been studied. The initiation reaction requires the parental protein p3 but not an intact DNA molecule. Protein p3-containing fragments from the left- or right-hand DNA ends were active as template for formation of the initiation complex provided they had a minimal size: a 26-base-pair-long fragment was active whereas a 10-base-pair-long one was essentially inactive. However, the activity of the latter was restored by ligation of an unspecific DNA sequence. phi 29 DNA internal fragments, as well as denatured phi 29 DNA, were inactive as template for the initiation reaction. The terminal protein-DNA complex isolated from Bacillus phage phi 15 was active in formation of the phi 29 p3-dAMP complex, whereas the protein-DNA complex isolated from Bacillus phage GA-1 or from the pneumococcal phage Cp-1, both with a morphology similar to that of phage phi 29, as well as that obtained from adenovirus, were inactive.

Bacillus subtilis↗
PubMed1984

Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication.

The phage phi 29 protein p2, required for the formation of the protein p3-dAMP initiation complex, has been purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of molecular weight 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when phi 29 DNA-protein p3 was used as template. In addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 mutant sus2-infected Bacillus subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of phi 29 DNA-protein p3 as template. However, when purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the phi 29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage phi 29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.

Bacillus subtilis↗
PubMed1984

Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.

Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3.

Bacillus subtilis↗
PubMed1983

Initiation of phage phi 29 DNA replication by the terminal protein modified at the carboxyl end.

A mutant at the carboxyl end of the terminal protein, p3, of phage phi 29 DNA has been constructed by inserting an containing the stop translation codon TGA in the three possible reading frames, immediately downstream of a phage phi 29 DNA fragment coding for all but the last five amino acids of protein p3. The activity in the formation of the p3-dAMP initiation complex in vitro of this mutant as well as another one previously isolated, also mutated at the carboxyl end, have been tested. The results obtained suggest that an intact carboxyl end in the phage phi 29 terminal protein is essential for its normal primer function in DNA replication.

Bacillus subtilis↗
PubMed1983

Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex.

To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in phi29 DNA replication, extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex. Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex, suggesting that these proteins are not involved in the initiation of phi29 DNA replication. ddATP or aphidicolin, inhibitors of DNA chain elongation, had also no effect on the formation of the initiation complex.

Adenosine Diphosphate↗
PubMed1983

Assembly of the tail protein of the Bacillus subtilis phage phi 29.

By in vitro complementation we have determined that gene 13 product functions during phi 29 morphogenesis after the formation of 11- particles, specifically in the functional assembly of the tail protein, p9. Protein p9 from 8- but not from 8-13- extracts assembles in vitro into either 11-13- or 12-13- particles. The action of gene 13 product on p9 for its correct assembly has to take place in vivo; no complementation of 12-13- and 9- lysates occurs in vitro. Protein p9, isolated from phi 29-infected cells, copurifies with the 13+ activity and it is present both in 13+ and 13- extracts as an aggregate with dimensions similar to those of the tail assembled in mature phage.

Bacillus subtilis↗
PubMed1983

Projections of the caudate nucleus in cats and kittens.

Small electrolytic lesions were placed on the head of the caudate nucleus of kittens between 8 and 21 days postnatally and in adult cats. Coronal sections of the whole brain were studied under the light microscope with the Fink and Heimer staining method. Degenerating axon terminals could be recognized after a survival period of 30 h for kittens and 8 days for adults, as dark spots and broken fibers. The results indicate extensive degenerative lesions at both diencephalic and brain stem levels. In general the degenerative lesions were denser in kittens when compared with adult animals. The results of this study suggest that the caudate nucleus of immature animals could be more sensitive to the lesions occurring during the time of early synaptogenesis. Moreover, the caudate nucleus could be an important point of convergence of descending pathways exerting a diffuse modulatory action upon several brain structures related to movement.

Animals↗
PubMed1983

Projections of the amygdala in rats during development.

Unilateral electrolytic lesions of the amygdaloid nucleus were made in rats during development to study the timing of sequential connectivity. The patterns of neural degeneration were detected by the Fink-Heimer technique and light microscopy. The main projections were to the accumbens, caudate, septal area, habenula, hypothalamus, cingulum, thalamus, and mesencephalon. In all cases degenerated fibers were progressively more apparent with increasing age. A clear and consistent pattern of degeneration was first observed at 30 days postnatally. Because the amygdala is widely connected with numerous limbostriatal and other CNS structures, the data presented favor the possibility that the amygdaloid complex can be a crucial region for the triggering of patterns of endocrine and autonomic, as well as somatic responses.

Amygdala↗