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Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 271 records · Page 15Linked to original sources
PubMed1982

High level synthesis in Escherichia coli of the Bacillus subtilis phage phi 29 proteins p3 and p4 under the control of phage lambda PL promoter.

The Hind III G fragment from the Bacillus subtilis phage phi 29 DNA, inserted downstream from the bacteriophage lambda promoter PL carried by a pBR322 derivative plasmid (pPLc28), directed the synthesis in E. coli of two proteins of apparent molecular weight 27500 and 12500. With the use of the recombinants obtained with the DNA from mutants sus3(91) and sus4(56), the two proteins were identified as a modified p3 (p3'), the protein covalently linked to the 5' ends of phi 29 DNA, and p4, responsible for the phi 29 late transcription, respectively. Under the best conditions used, proteins p4 and p3' were produced in E. coli from the cloned DNA fragments in an amount corresponding to approximately 30% and 6% of total de novo protein synthesis, respectively.

Bacillus subtilis↗
PubMed1982

Nucleotide sequence of the early genes 3 and 4 of bacteriophage phi 29.

The nucleotide sequence of an early region of the phi 29 genome has been determined. The sequenced region includes genes 3 and 4, which code for the protein covalently linked to the 5' ends of phi 29 DNA and the protein involved in the control of late transcription, respectively. The position and nature of the mutations of mutants sus3(91) and sus4(56) has also been determined.

Amino Acid Sequence↗
PubMed1982

Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.

Incubation of extracts of phi 29-infected Bacillus subtilis with [alpha-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the phi 29 DNA-protein p3 linkage is hydrolyzed released 5'-dAMP. The reaction with [alpha-32P]dATP was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex; no reaction took place with proteinase K-treated phi29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5'-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 microM [alpha-32P]dATP can be elongated by addition of 40 microM dNTPs. Treatment with piperidine of the product elongated in the presence of 2',3'-dideoxycytidine 5'-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.

Bacillus subtilis↗
PubMed1981

Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29.

Phage phi 29 DNA cannot be phosphorylated with polynucleotide kinase and [gamma-32P]ATP because of the presence of a viral protein covalently linked to the 5' termini. The 5' ends can, however, be made susceptible to phosphorylation by treatment with alkali and alkaline phosphatase. Restriction fragments Hpa II C and Hpa II F, corresponding to the right and left ends of phi 29 DNA, respectively, were labeled at the 5' ends with polynucleotide kinase and [gamma-32P]ATP or at the 3' ends with terminal transferase and [alpha-32P]ATP or [alpha-32P]cordycepin 5'-triphosphate. After a secondary cleavage of the labeled fragments, the sequence of the first 150-180 nucleotides at the termini of phi 29 DNA was determined by the method of Maxam and Gilbert. The ends of phi 29 DNA are flush, and a six-nucleotides-long inverted terminal repetition was found. The functional implications of the sequences determined are discussed.

Alkaline Phosphatase↗
PubMed1981

Purification, properties and assembly of the neck-appendage protein of the Bacillus subtilis phage phi 29.

The purification of the neck appendage protein of phi 29, p12*, which is involved in the adsorption of the phage to Bacillus subtilis, is described. The purified native protein is in a dimeric form and can be assembled, in vitro, onto purified 12- particles that lack the neck appendages, suggesting that the incorporation of p12* to the rest of the phage structure is a self-assembly process. The assembly of protein p12* in vitro follows cooperative kinetics and it occurs with an efficiency of about 4%.

Bacillus subtilis↗
PubMed1981

Adsorption of bacteriophage phi 29 to Bacillus subtilis through the neck appendages of the viral particle.

Phage phi 29 particles produced under restrictive conditions by mutants in gene 12 have normal amounts of all of the structural proteins except the appendage protein, p12*, which is missing. These particles are not infective and do not adsorb to Bacillus subtilis cells. By in vitro complementation of 12- particles with extracts containing protein p12* or with purified protein p12*, the defective particles could bind the appendage protein and become infective and able to adsorb to bacteria. Therefore, the neck appendages of phage phi 29, formed by protein p12*, are involved in the interaction of the phage with the cell wall receptors. Protein p12*, purified in its native state, competed with wild-type phage for adsorption to bacteria. Also, protein p12* could displace adsorbed phage from bacteria. Since the displaced phage was infective, protein p12* does not seem to be modified after phage adsorption.

Adsorption↗
PubMed1980

Bacteriophage phi 29 infection of Bacillus subtilis minicells.

Bacteriophage phi 29 can infect B. subtilis minicells and synthesize all the phage-coded proteins detected in ultraviolet irradiated-infected B. subtilis cells. Synthesis of phage unit-length DNA has been obtained after infection of minicells with phi 29. The DNA can be encapsulated in particles with a sedimentation rate similar to that of phage phi 29 produced in B. subtilis cells. The particles produced in minicells can be adsorbed to B. subtilis cells, but infectivity has not been demonstrated because of the very low burst-size obtained.

Bacillus subtilis↗
PubMed1980

Liver ultrastructure during acute stress.

An ultrastructural, morphologic and histochemical study was made on the livers of rats exposed to eight different acute stressors: fasting, cortisol injecions, reserpine injections, restraint, spinal cord transection, immersion in hot water, exposure to cold and forced muscular exercise in a revolving drum. After 48 hours of exposure to stress, electron microscopy of the liver revealed rough endoplasmic reticulum fragmentation and dilatation, glycogen depletion, and mitochondrial enlargment. The most striking change, however, was an increase in the number and size of autophagic vacuoles which were limited by single or multiple membranes. A cytochemical study revealed that in the former case, the vacuolar membranes did not show a glucose-6-phosphatase positive reaction, whereas they did in the latter case. The vacuoles contained acid phosphatase positive material as well as organelles in various stages of degradation. Following exposure to most of the stressors, a marked increase of plasma corticosterone was noted, with a lowered rectal temperature and the appearance of the typical stress triad (adrenal hypertrophy, thymicolymphatic involution and gastrointestinal ulcers). The severity of the morphologic changes appeared to parallel the degree of hypothermia caused by the stressor. The results suggest that autophagy in the liver may be an adaptive response to stressors at the subcellular level.

Animals↗
PubMed1980

Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP.

To investigate the role of protein p3 in bacteriophage phi 29 initiation of replication, we have studied the nature of the covalent linkage between protein p3 and phi 29 DNA. The protein-DNA compound was digested with micrococcal nuclease and pronase resulting in a nucleotidyl-peptide that was further digested by alkaline phosphatase and snake venom phosphodiesterase yielding 5'-dAMP. The DNA-protein linkage is sensitive to alkali. Treatment of the nucleotidyl-peptide with 0.1 M NaOH at 37 degrees C for 3 hr after phosphatase digestion released 5'-dAMP. Hydrolysis of the nucleotidyl-peptide with 5.8 M HCl at 110 degrees C for 90 min yielded O-phosphoserine. These results, together with the sensitivity of the DNA-protein linkage to snake venom phosphodiesterase and its resistance to hydroxylamine, indicate that protein p3 is covalently linked to phi 29 DNA through a phosphoester bond between L-serine and 5'-dAMP, namely a O,5'-deoxyadenylyl-L-serine bond.

Adenosine Monophosphate↗
PubMed1980

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.

Bacillus subtilis↗
PubMed1980

Effects of early undernutrition on dendritic spines of cortical pyramidal cells in the rat.

Restricted food intake in male rats between birth and 20 days of age reduced the density of spines of layer V pyramidal cells in the frontal, parietal and occipital cortices. The loss of dendritic spines was more marked in pyramidal cell areas receiving projections from nonspecific multisynaptic systems than in cell areas associated with specific afferent systems or in areas receiving callosal projections. It was suggested that the differential effect of undernutrition in nonspecific, specific and callosal afferent projection areas of the pyramidal cortex might be related to the impairment of brain integrative functions reported to follow perinatal undernourishment.

Animals↗
PubMed1980

Cell surface adenosine 5'-triphosphatase as an in vitro marker of the lineage and cytodifferentiation of oncogenic epithelial cells from rat liver parenchyma.

The relationship between the oncogenicity and the surface properties of cultured liver epithelial cells has been studied with the newborn Wistar rat-derived euploid line, RL34, and its heteroploid variants. An oncogenic variant, RL34HT, appeared to be more functionally active than its nononcogenic counterparts with respect to cell surface adenosine 5'-triphosphatase (ecto-ATPase) as well as to cytoplasmic enzymes such as tyrosine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase. The cell surface of RL34HT was distinguished from those of nononcogenic and marginally oncogenic cell populations by the presence of abundant microvilli and by the absence of large external transformation-sensitive protein (fibronectin). High-Km and high-Vmax Ca2+-Mg2+ -ecto-ATPase was found in RL34HT. All nononcogenic cell lines had a flat granular surface membrane with high levels of fibronectin and also exhibited ecto-ATPase activity with low Km and low Vmax. When RL34HT was grown in dibutyryl cyclic adenosine 3',5'-monophosphate and theophylline, the external cell surface was partially restored to the polypeptide compositions of RL34, and there was an increase in Vmax of ecto-ATPase without a change in Km. The high-Km ecto-ATPase may be a useful indicator reflecting the lineage and cytodifferentiation of oncogenic liver epithelial cells, since it is also known to be localized at the bile canalicular microvilli of normal adult hepatocytes.

Adenosine Triphosphatases↗
PubMed1980

Effects of neonatal exposure to paint thinner on the development of swimming in rats.

Rats were exposed to paint thinner twice a day for a period of 10 minutes on Days 1 through 30 of postnatal life. The subsequent effects upon physical development, swimming ability and escape latency from water were evaluated. Maturation of swimming behavior and general physical development were delayed about 2-4 days in the experimental animals compared with non-exposed littermate controls. The results of these experiments suggest that exposure to this organic solvent during the early postnatal period interferes with the development of the cortico-subcortical neural structures underlying swimming and locomotion.

Animals↗
PubMed1979

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.

Bacillus subtilis↗