Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Expression and characterization of the tau subunit of phosphorylase kinase

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.     

The major type-1 protein phosphatase catalytic subunits are the same gene products in rabbit skeletal muscle and rabbit liver

The catalytic subunit of protein phosphatase-1 (PP1) isolated from rabbit liver had the same electrophoretic mobility as, and yielded peptide maps identical to those of the 33 kDa form of rabbit skeletal muscle PP1. The predicted amino-acid sequences of PP1 obtained from three rabbit liver cDNA clones were identical to that of PP1 alpha from rabbit skeletal muscle. These findings suggest that the distinctive substrate specificities and regulatory properties of hepatic and skeletal muscle type-1 protein phosphatases are not conferred by the catalytic subunits themselves, but by regulatory subunits that are complexed to the catalytic subunits in vivo.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum

The complete nucleotide sequence of the celH gene of Clostridium thermocellum was determined. The open reading frame extended over 2.7-kb DNA fragment and encoded a 900-amino acid (aa) protein (Mr 102,301) which hydrolyzes carboxymethylcellulose, p-nitrophenyl-beta-D-cellobioside, methylumbelliferyl- beta-D-cellobioside, barley beta-glucan, and larchwood xylan. The N terminus showed a typical signal peptide, and a cleavage site after Ser44 was predicted. Two Pro-Thr-Ser-rich regions divided the protein into three approximately equal domains. The central 328-aa region was similar to the N-terminal part, carrying the active site, of C. thermocellum endoglucanase E (EGE; 30.2%). The C-terminal region ended with two conserved 24-aa stretches showing close similarity with those previously described in EGA, EGB, EGD, EGE, EGX, and xylanase from C. thermocellum. Deletions of celH removing up to 327 codons from the 5' end and up to 245 codons from the 3' end of the coding sequence did not affect enzyme activity, confirming that the central domain was indeed responsible for catalytic activity. Production of truncated EGH in Escherichia coli was increased up to 120-fold by fusing fragments containing the 3' portion of the gene with the start of lacZ' present in pTZ19R.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.