Search PubMed⌕ Search

Biomedical subjects

F Brunel

Publications and source records attributed to F Brunel.

48 records · Page 3Linked to original sources
PubMed1982

Cloning and characterization of a genomic DNA fragment carrying the basic copy of the gene coding for variant surface antigen 118 of Trypanosoma brucei.

It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.

Animals↗
PubMed1981

Cloning of bacteriophage T5 DNA fragments. II. Isolation of recombinants carrying T5 PstI fragments.

The adjacent PstI-J, I and G fragments of the phage T5 DNA molecule (4.4, 4.6 and 7.2 kb, respectively) have been cloned in plasmid pBR322 and their locations verified by Southern blot analysis. The PstI I and G fragments overlap the previously cloned HindIII-P and G fragments and like those, contain no known genetic markers. In addition, one of the 12 newly isolated T5 mutants maps in this PstI-IG region. Thus, the size of the "empty" region between genes D15 and D17, which we have previously observed on the genetic map, extends to at least 11.8 kb. In contrast, the PstI-J fragment carried part of the D12 gene and the intact D14 and D15 genes. This clone is of particular interest since the D15 gene product is a nuclease and is responsible for the positive control of late gene transcription. The orientation of these genes relative to the T5 DNA molecule has been determined by a combination of restriction, deletion and complementation analyses.

Chromosome Mapping↗
PubMed1981

Cloning of bacteriophage T5 DNA fragments. III. Expression in Escherichia coli mini-cells.

Use has been made of the mini-cell system to study polypeptide synthesis from cloned EcoRI, HindIII and PstI fragments of T5 DNA. The correlation of certain gene products with known genes has been established, as well as the physical mapping of genes not yet identified genetically. In some cases, it has been possible to demonstrate the presence of T5 promoters on the cloned DNA fragments. The design of experiments to avoid certain artifacts inherent in the use of the mini-cell system is discussed.

Bacteriophage lambda↗
PubMed1980

Maxi-circles and mini-circles in kinetoplast DNA from trypanosoma cruzi.

Maxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by electron microscopy. Long loops up to 10 micrometer were present, extending from the network rim; these are considered typical of maxi-circles. The presence of maxi-circles was proven by digestion of kinetoplast DNA with restriction endonucleases and S1 nuclease. This released a minor DNA component, detectable by agarose gel electrophoresis, which hybridized to maxi-circle DNA from Trypanosoma brucei. The molecular weight of the linearized maxi-circle of Trypanosoma cruzi is 26 . 10(6), as judged from its electrophoretic mobility in 0.6% agarose. Our restriction enzyme analysis of the mini-circles of Trypanosoma cruzi has confirmed their sequence heterogeneity and internally-repeated structure. We have found that more than 90% of the mini-circles are cut into 1/4 length molecules by endonuclease TaqI. Denaturation and renaturation of mini-circles, cut once with endonuclease MboI, mainly yields linear and circular molecules with single-stranded eyes and tails in electron micrographs. This shows that 1/4 repeats contain sub-segments in which sequence divergence is extensive. Our EcoRI and HapII digests differ in fragment size distribution from those previously reported. This suggests that this distribution may not be a stable characteristic of the Y strain.

Animals↗
PubMed1980

Cloning and expression of Trypanosoma brucei kinetoplast DNA in Escherichia coli.

The kinetoplast DNA of Trypanosoma brucei is made of two components: mini-circles (1 kb, 90% of total kDNA) and maxi-circles (20 kb, 10%) of total kDNA). These are interlocked to form a network of about 10 000 kb. In order to analyse the components of such a network structure, we have cloned individual mini-circle molecules and two of the three EcoRI maxi-circle fragments in E. coli. Cloned mini-circles are somewhat heterogeneous in size and their restriction patterns are completely different. Despite this heterogeneity all are found to contain a homologous region(s) defined by DNA/DNA hybridization. The maxi-circles probably correspond to the mitochondrial DNA of other organisms and, in contrast to mini-circles, do not show sequence heterogeneity. One of the two cloned maxi-circle EcoRI fragments is able to direct the synthesis of two polypeptides of 10 300 and 13 500 daltons in E. coli mini-cells. Detailed analysis of this phenomenon shows that both structural genes and promoter(s) are located within the cloned maxi-circle fragment.

Animals↗
PubMed1979

Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES.

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.

Chromosome Mapping↗
PubMed1979

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.

Bacteriophage lambda↗
PubMed1979

Restriction insensitivity in bacteriophage T5 I. Genetic characterization of mutants sensitive to EcoRI restriction.

Unmodified bacteriophage T5 is able to grow normally on bacterial hosts carrying three different Escherichia coli restriction systems, EcoK, EcoPI, and EcoRI. Under the same conditions, the plating efficiency of bacteriophage gamma is less than 10(-9). At least in the case of EcoRI, this lack of in vivo restriction is not due to lack of restriction sites on the T5 DNA molecule. These observations suggest that bacteriophage T5 specifies one or more restriction protection systems. Mutants (ris) of T5 have been isolated which confer sensitivity to EcoRI restriction but not to EcoK or EcoPI. The mutations are located in the pre-early region of the genetic map but are too far apart to be alleles of a single gene. Complementation studies show that the ris mutants can be helped to grow on the EcoRI-restricting host by coinfection with T5+. This result provides evidence for a restriction protection function but does not necessarily show that the ris mutants are defective in such a system.

Coliphages↗
PubMed1979

Restriction insensitivity in bacteriophage T5. II. Lack of EcoRI modification in T5+ and T5ris mutants.

Neither bacteriophage T5+ nor its EcoRI-sensitive ris mutants became modified during growth on an EcoRI-modifying host. For this reason, the rare ris plaques able to grow on the EcoRI-modifying host were always due to revertant phage rather than to modified ris mutants. The ris mutations resulted in the creation of new EcoRI cleavage sites in the terminally repetitious first-step transfer DNA, and analysis of T5 ris revertants showed loss of these sites and restoration of the wild-type restriction pattern. Natural EcoRI sites present in the second-step transfer DNA were never lost in T5ris revertants, indicating that these are irrelevant to in vivo restriction and are protected during growth on the restricting host.

Coliphages↗