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M Heusterspreute

Publications and source records attributed to M Heusterspreute.

27 records · Page 2Linked to original sources
PubMed1985

Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon.

We present the nucleotide sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the lambda gal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in lambda gal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage lambda. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the galK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

Adenosine Triphosphate↗
PubMed1985

Vectors with restriction-site banks. III. Escherichia coli-Saccharomyces cerevisiae shuttle vectors.

The bank of unique restriction sites present in plasmid pJRD158 has been incorporated into new vectors carrying selective markers and replicons derived from commonly used Escherichia coli-Saccharomyces cerevisiae shuttle vectors pJDB207 and YRp7. The new vectors pMH158 and pJO158 have 21 and 23 unique restriction sites, respectively, and their complete DNA sequences are known.

Base Sequence↗
PubMed1984

Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites.

A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles. Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes. The plasmid has the additional advantages of very high copy number and altered incompatibility. The latter permits it to be stably maintained in the same host as pBR322.

Base Sequence↗
PubMed1984

Restriction site bank vectors. II. DNA sequence analysis of plasmid pJRD158.

pJRD158 is a small plasmid vector (3903 bp) derived from pBR327 and specifying resistance to ampicillin and tetracycline. It contains 28 unique restriction sites (and 4 nonunique restriction sites) that can be used for cloning. The DNA sequence and computer-assisted restriction site analysis of pJRD158 are reported. Evidence is also presented that suggests a 2-bp revision of the DNA sequence of pBR322 in the RNA primer region.

Ampicillin↗
PubMed1984

Expression of galactokinase as a fusion protein in Escherichia coli and Saccharomyces cerevisiae.

Plasmids are described that allow fusions between the Escherichia coli galK gene (coding for galactokinase) and any gene of interest. An example is given in which a galK gene, lacking the normal initiator methionine codon, is fused to various segments of the 5' end of the tetR gene of pBR322. The resulting plasmids complemented an E. coli galK mutant, and galactokinase activity was retained despite the addition of up to 250 foreign amino acids to the amino-terminus of the galactokinase polypeptide. In a second experiment, the galK gene was fused to the LEU2 gene of Saccharomyces cerevisiae. The resulting plasmid was able to complement a yeast GAL1-mutant and galactokinase synthesis in yeast was controlled, via the LEU2 regulatory system, by the levels of leucine and threonine in the growth medium. The galK fusion plasmids should facilitate analysis of the control systems of a wide variety of genes in different organisms.

Base Sequence↗
PubMed1983

Gene rearrangements leading to the expression of an insertion-inactivated tetracycline resistance gene in pBR322.

Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene. The corresponding clones revert to tetracycline resistance at a low frequency. Such reversion is caused by gene rearrangement within the plasmids. DNA sequence analysis reveals three classes of revertants. The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene. The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule. The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter. In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact. However, it removes the promoter of the RNA primer, the latter now being read from the cloned material. The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter. The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator. The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription.

Base Sequence↗
PubMed1983

A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322.

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.

Chromosome Deletion↗