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J C Zabala

Publications and source records attributed to J C Zabala.

30 records · Page 2Linked to original sources
PubMed1993

Incorporation of tubulin subunits into dimers requires GTP hydrolysis.

A toroid multisubunit complex of 800-900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different alpha- and beta-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.

Adenosine Triphosphate↗
PubMed1992

Tubulin dimer formation via the release of alpha- and beta-tubulin monomers from multimolecular complexes.

The functional subunit of microtubules is a heterodimer consisting of alpha- and beta-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse alpha-tubulin isotypes involves a multimolecular complex. The synthesis of mouse beta-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized alpha- and beta-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers.

Cell-Free System↗
PubMed1987

Transposition of IS91 does not generate a target duplication.

We determined the DNA sequences surrounding the junctions of IS91 in two insertion derivatives: pSU234 (pACYC184::IS91) and pSU240 (pBR322::IS91). The termini of IS91 consist of two imperfect inverted repeats eight base pairs long. Their sequence is 5'-TCGAGTAGG. . . CCTATCGA-3'. Insertion of IS91 did not generate direct repetitions in the target DNAs.

Base Sequence↗
PubMed1985

Purification of alpha-hemolysin from an overproducing E. coli strain.

The genetic determinant of the alpha-hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions. alpha-Hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified alpha-hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the alpha-hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.

Bacterial Proteins↗
PubMed1984

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements.

A revised physical map of the alpha-haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.

Bacterial Proteins↗
PubMed1984

Characterization of the new insertion sequence IS91 from an alpha-hemolysin plasmid of Escherichia coli.

IS91 is a 1.85 kb insertion sequence originally resident in the alpha-hemolytic plasmid pSU233. The element was transposed sequentially from this plasmid to pACYC184, to R388, and to pBR322. Both cointegrates and simple insertions of the element were obtained. A detailed restriction enzyme map of the element is presented. This does not bear any relationship to the maps of previously described insertion sequences. Furthermore, hybridization between these sequences and IS91 could not be demonstrated. Deletion derivatives of IS91 were constructed which are unable to transpose. However, their transposition can be complemented in trans by wild-type elements. One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5. When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates. Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition.

Bacterial Proteins↗
PubMed1983

Relationship of the replication and incompatibility genes of an IncFVI haemolysin plasmid with other F-like haemolysin plasmids.

The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12.6 kb plasmid, denominated pSU503. Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233). Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (less than 70%) with any of the haemolysin-determining plasmids with which it is compatible. Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons.

Base Sequence↗
PubMed1983

Hemolysis determinant common to Escherichia coli strains of different O serotypes and origins.

Ten Escherichia coli hemolytic strains, isolated from various types of human infection and belonging to at least six different O serotypes were analyzed. In all these strains, our results are consistent with the hly genetic determinant being located on the bacterial chromosome. An hly-specific probe (plasmid pAN215) was used to detect homologous fragments in the strains analyzed by the Southern blotting technique. It was found that all 10 strains contain sequences that hybridize against the probe (greater than 85% homology) and thus contain hly genetic determinants very similar to those found on Hly plasmids. These results strongly suggest that there is a unique ubiquitous genetic determinant responsible for the hemolytic phenotype in E. coli.

Chromosomes, Bacterial↗
PubMed1982

Several copies of the same insertion sequence are present in alpha-hemolytic plasmids belonging to four different incompatibility groups.

Alpha-hemolytic plasmids belonging to four different incompatibility groups contained several copies (two to six) of the same DNA sequence. This sequence was capable of illegitimate recombination and thus behaved as an insertion sequence element. Its size (1.9 kilobases) and restriction enzyme cleavage map suggested that it was different from previously described insertion sequence elements.

Conjugation, Genetic↗