Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine αs1-casein-CAT gene

Two αs1-casein/chloramphenicol acetyltransferase (CAT) gene constructs overlapping by 3.0kb were constructed and co-injected into murine zygotes. In 9 of 10 lines of transgenic mice obtained, based on analysis of structure and expression of the transgene, accurate extrachromosomal homologous recombination (ECR) between the two overlapping DNA fragments was found. Different levels of CAT activity were detected in milk from these lines. The highest CAT activity was about 25-50μg/mL milk. In some mice. CAT activity was found in salvia gland, thymus and spleen extracts. The high frequency and accuracy of ECR reported here will be applicable in the experimental manipulation for generation of relatively large transgene.     

Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine αs1-casein-CAT gene

Two αs1-casein/chloramphenicol acetyltransferase (CAT) gene constructs overlapping by 3.0kb were constructed and co-injected into murine zygotes. In 9 of 10 lines of transgenic mice obtained, based on analysis of structure and expression of the transgene, accurate extrachromosomal homologous recombination (ECR) between the two overlapping DNA fragments was found. Different levels of CAT activity were detected in milk from these lines. The highest CAT activity was about 25-50μg/mL milk. In some mice. CAT activity was found in salvia gland, thymus and spleen extracts. The high frequency and accuracy of ECR reported here will be applicable in the experimental manipulation for generation of relatively large transgene.     

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.     

Unstable, non-mutational expression of resistance to the thymidine analogue, trifluorothymidine in CHO cells.

In Saccharomyces cerevisiae, methyl methanesulphonate and diepoxybutane produced efficiently lethal, as well as mutagenic, damage in nuclear DNA. However, in the same conditions, these agents did not induce cytoplasmic petite mutations and poorly induced point mutations (resistance to erythromycin and chloramphenicol) in mitochondrial DNA. Possible reasons for these differences are discussed.     

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.     

Study of the maturation of 5 s RNA precursors in Escherichia coli

The existence of three precursors to 5 s RNA's (p5 s RNA's) has been confirmed. p5 s RNA's and mature 5 s RNA have different 5′-terminal sequences and produce the following 5′-terminal oligonucleotides: p5 s RNA-I:pAUUUG; p5 s RNA-II:pUUUG; p5 s RNA-III:pUUG; 5 s RNA:pUG. The results of experiments on pulse-labelled cells treated with actinomycin D, on chloramphenicol-inhibited cells, on various ribosome assembly-defective mutants and on the state of 5 s RNA in polysomes after a short labelling period, support the following conclusions. (1) p5 s RNA-I is the first identifiable precursor which appears during a pulse. (2) The amount of p5 s RNA-II, relative to those of the other p5 s RNA's, is very low at all times during pulse-chase experiments. On the contrary, it becomes significant during chloramphenicol inhibition and in one assembly-defective mutant under non-permissive conditions. (3) The maturation steps which lead to p5 s RNA-III and 5 s RNA normally occur after binding to 50 s subunit precursor particles and are, consequently, dependent upon protein synthesis. (4) The transition from p5 s RNA-III to 5 s RNA is a slow process, which is neither dependent upon nor required for the proper functioning of 50 s subunits in protein synthesis.     

A vector for recombinant DNA in Staphylococcus aureus

Staphylococcal plasmids pS194 and pSC194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant DNA, since they each carry one single recipient site for endonuclease EcoRI. Hybrid DNA does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by EcoRI. A chloramphenicol marker present in pSC194 was used for positive hybrid selection. Hybrid plasmids generated by joining pSC194 with one or more of the four EcoRI fragments of the large (18.1-10(6) daltons) staphylococcal plasmid pI258 were constructed and permitted us to develop a physical map for pI258.     

Coupling of processing of alpha-glucosyl transferase mRNA with translation.

Bacteriophage T4 α-glucosyl transferase mRNA is made as a polycistronic 21S molecule that is processed during normal infection to the commonly found 14.5S species. By using antibiotic inhibitors of protein synthesis, it is possible to distinguish two steps involved in the processing of the 21S polycistronic α-gt mRNA in T4-infected $\text{Escherichia coli}$. There is an initial cleavage to an 18S molecule that does not require protein synthesis. However, the next step, the conversion of the 18S into the 14.5S molecule, requires simultaneous protein synthesis.