David Zipser

Stabilization of a degradable protein by its overexpression in Escherichia coli

Synthesis of proteins in Escherichia coli using recombinant DNA methodology has become an important tool for isolating and studying proteins. However, the E. coli protein degradation systems can interfere with the expression of cloned genes. To examine the effect of protein degradation, we have cloned the X90 allele of the E. coli lacZ gene. The X90 allele, an ochre mutant, codes for beta-galactosidase lacking approx. 12 amino acids from the carboxyl terminus. The X90 protein is rapidly degraded in wild-type E. coli. Randomly sheared DNA fragments from lambda placZ-X90 were inserted into the EcoRI site of the plasmid pOP203-UV5-3, a derivative of pMB9 containing the lactose operator-promoter region. Recombinant plasmids that carry the lacZ-X90 gene were identified by the Lac+ phenotype of their transformants in an ochre-suppressor-containing host and the Lac- phenotype in Su degrees or supE hosts. One recombinant plasmid, p41, with an insert of 7.6 kb codes for the synthesis of the X90 promoter at a quantity equal to or greater than 50% of the total cellular protein of several strains. In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates. The surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.

Mapping of Restriction Sites in the Attachment Site Region of Bacteriophage Lambda

SummaryA fine structure map of theEcoRI fragment containing the lambda attachment-site region has been constructed. 38 different restriction endonucleases have been employed and 170 sites located in this fragment. In addition, sites in adjacent regions have been determined for several enzymes. Complete cleavage maps of the entire lambda genome have been obtained for endonucleasesBglII,BluI,KpnI,SacI,SacII,SalI andXbaI. The strategy employed for mapping included comparison of deletion and substitution mutants, analysis of mixed digests, and detailed analysis of subfragments.

The molecular cloning of the immunity gene of phage Mu

Abstract The 1000 base pair left-end Hin dIII fragment of phage Mu DNA was cloned in the Eco RI cleavage site of plasmid pMB9, using poly(dA·dT) linkers added with terminal transferase. These plasmids conferred a high level of Mu immunity on their hosts. When the plasmids were segregated into minicells, a 25 000 molecular weight protein correlated with immunity could be identified by labeling and gel electrophoresis.

Easy-to-use equipment for the accurate microinjection of nanoliter volumes into the nuclei of amphibian oocytes

Abstract Microinjection of Xenopus oocytes has become a valuable technique for studying many aspects of control of eucaryotic gene expression. Previously published methods for controlling and monitoring the volume of sample injected into oocytes are time consuming and tedious and require considerable expertise to obtain reproducible results. The construction and manner of usage of micropipets are the most critical parts of a successful injection technique. Methods for the construction of micropipets and ways to assess the accuracy and precision of the injection of nanoliter volumes into amphibian oocytes are described. Construction of pipets is done almost entirely using instruments that are relatively inexpensive and allow for a high degree of reproducibility. Micropipets made in this manner have several advantages: (1) They inflict little damage on the oocytes during the injection process; greater than 95% of the oocytes survive after microinjection. (2) Oocytes do not require defolliculation prior to microinjection. (3) One micropipet can be used to inject as many as 100 oocytes before needing to be refilled with sample and without acquiring a blockage. Using the microinjection techniques described, the accuracy of sample delivery was within 9% of the mean and the precision increased with increasing volume: the standard deviations of volumes in the 5-nl range were within 9% of the mean and volumes in the range 10–50 nl were within 4–6% of the mean.

Transfection of Escherichia coli by Mu DNA.

SummaryInfectivity of Mu DNA was demonstrated in Ca++-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.

Genetic analysis of the cloned genome of phage Mu

Abstract Randomly sheared fragments of Mu cts 62 DNA have been cloned into the Eco RI cleavage site of plasmid p0P203(UV-5)-3, a derivative of pMB9 containing the lactose operator promoter, using poly (dA · dT) tailing with terminal transferase. The extent of Mu DNA carried by the recombinant plasmids was determined genetically by crosses with known markers in essential genes of Mu in rec + backgrounds. Plasmids which rescued essential genes were transformed to rec − backgrounds to check for expression of the Mu genes. Plasmids were found which rescued and complemented 23 of the 24 essential genes of Mu. Many of these plasmids carried overlapping segments of the Mu genome. The plasmid library was also screened for clones containing the nonessential genes gin and mom . Clones carrying gene A were not found in the initial screening, however, several clones which were resistant to infection by Mu due to expression of the Mu repressor were able to rescue and complement a D108 cts -Mu Ats hybrid phage at 43°.

Identification of the gin protein of bacteriophage Mu

Abstract The Mu gin function catalyzes a site-specific intramolecular recombination event which leads to the inversion of the Mu G segment. Here we report the subcloning of the gin gene next to the strong lac promoter carried on a multicopy plasmid. Comparison of the protein patterns of Gin + recombinant plasmids with the protein patterns of Gin − plasmids derived by in vitro mutagenesis allowed identification of the gin product as a protein of molecular weight 21,500.

Mispair correction in nucleic acids with alternate forms

The effect of base mispair correction on nucleic acids able to alternate between different paired forms was studied by computer simulation. Several phenomena were observed and are described. They include the accumulation of internal homologies, the existence of nonrectifiable molecules which are perpetually corrected, and of hypervariable sequences in which a large proportion of the nucleotides can be altered by correction. The suggestion is made that hypervariability could account for antibody diversity and perhaps play other significant roles in development and evolution.

Random Nonhomologous Recombination

Summary The totally random insertion of phage Mu-1 into the Escherichia coli chromosome is described. Its significance for tumor viruses and genetic engineering is discussed.