We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.
The mating‐type region of fission yeast consists of three components, mat1, mat2‐P and mat3‐M, each separated by 15 kb. Cell‐type is determined by the alternate allele present at mat1, either P in an h+ or M in an h‐ cell. mat2‐P and mat3‐M serve as donors of information that is transposed to mat1 during a switch of mating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating‐type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at mat2‐P and mat3‐M but absent at mat1 (H3; 57 bp), and may be involved in transcriptional repression of these cassettes. mat1‐P and mat1‐M each encode two genes (Pc; 118 amino acids, Pi; 159 amino acids, Mc; 181 amino acids and Mi; 42 amino acids). Introduction of opal or frame‐shift mutations into the open‐reading‐frame of each gene revealed that Pc and Mc are necessary and sufficient for mating and confer an h+ or h‐ mating type respectively. All four genes are required for meiotic competence in an h+/h‐ diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen‐free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.
Aminoglycoside antibiotics in Escherichia coli and yeast can cause ribosomes to read through stop codons during translation. This can result in the phenotypic suppression of nonsense mutations. We show here for the first time that the aminoglycosides G-418 and paromomycin have similar effects in monkey (COS-7) cells in vivo. Suppression of an amber mutation (TAG) by aminoglycosides can restore the activity of a mutant gene transfected into COS-7 cells to almost 20% of wild type levels.
G protein-coupled receptors (GPCRs) are the largest family of genes in animal genomes and represent more than 2% of genes in humans and C. elegans. These evolutionarily conserved seven-transmembrane proteins transduce a diverse range of signals. In view of their pivotal role in cell signaling, it is perhaps surprising that decades of genetic analysis in C. elegans, and recent genome-wide RNAi screens, have identified very few GPCR mutants. Therefore, we screened all GPCRs predicted to bind either small-molecule neurotransmitters or neuropeptides by using RNAi and quantitative behavioral assays. This shows that C16D6.2, C25G6.5, C26F1.6, F35G8.1, F41E7.3, and F59C12.2 are likely to be involved in reproduction, whereas C15B12.5, C10C6.2, C24A8.4, F15A8.5, F59D12.1, T02E9.1, and T05A1.1 have a role in locomotion. Gene deletions for F35G8.1 and T05A1.1 resulted in the same phenotype as that seen with RNAi. As some GPCRs may be resistant to RNAi, or may result in abnormalities not screened for here, the actual proportion of nonredundant receptors with an assayable function is probably greater. Strikingly, most phenotypes were observed for NPY-like receptors that may bind neuropeptides. This is consistent with the known actions of neuropeptides on the body wall muscle and reproductive tract in nematodes.
Introduction Circulating tumor cells (CTCs) are detectable in most cancer patients and they can meet an existing medical need to monitor cancer patients during a course of treatment and to help determine recurrent disease. CTCs are rarely found in the blood of cancer patients and enrichment is necessary for sensitive CTC detection. Most CTC enrichment technologies are anti-EpCAM antibody based even though CTC identification criteria are cytokeratin positive (CK + ), CD45 negative (CD45 - ) and 4'6-diamidino-2-phenylindole (nuclear stain) positive (DAPI + ). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or a combination of anti-CK and anti-EpCAM antibodies. Methods Blood samples from 49 patients with metastatic breast cancer were processed using the CellSearch™ system (Veridex, LLC, Raritan, NJ, USA), in parallel with our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol ® system (Genetix USA Inc, San Jose, CA, USA) was used for automated cell image capture and analysis of CTCs on glass slides. Results Our method has the capability to enrich three types of CTCs including CK + &EpCAM + , CK + &EpCAM -/low , and CK -/low &EpCAM + cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearch™ system in the total of 49 breast cancer patients. Our method detected 15 to 111% more CTCs than the CellSearch™ method in patients with higher CTC counts (>20 CTCs per 7.5 ml of blood). The three fluorescent and brightfield images from the Ariol ® system reduced the number of false-positive CTC events according to the established CTC criteria. Conclusion Our data indicate that the tumor-specific intracellular CK marker could be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol ® system reduced false-positive CTC events.
Resonance Raman spectra are reported for recombinant horseradish peroxidase C (HRP-C*) and three protein variants prepared by in vitro refolding after Escherichia coli expression. The spectra of their FeII and FeIII forms and of their complexes with benzohydroxamic acid (BHA) were recorded at neutral pH. The residues mutated were on the distal [Phe41-->Trp or Val (F41W, F41V) and Arg38-->Lys (R38K)] side of the heme. The spectra give information on the spin and ligation states via the frequencies of the core size marker bands. No detectable modification in the enzyme structure or in the heme group has been observed in the wild-type recombinant HRP-C*. The FeIII forms of both the recombinant and the plant proteins show the coexistence of a 5-(5-cHS) and a 6-coordinate high-spin (6-cHS) heme, characterized by the anomalous frequencies of certain bands, namely, v3 and v10, which we attribute to a different degree of distortion of the heme planarity with respect to other heme proteins and model compounds, resulting from external forces such as steric contacts within the protein. This effect is partially relieved upon complexation with BHA or as a result of mutation. F41W and F41V are characterized by an increase in a 6-cHS form at the expense of the 5-cHS species, and the R38K by an increase in both the 6-c high-(HS) and low-spin (LS) hemes. The 6-cHS and -LS species are characterized by normal core size marker band frequencies. The FeII-His RR band is at 243 cm-1 in HRP-C*, the high frequency value being due to hydrogen-bonding interactions between the proximal His170 N delta and the carboxylate acceptor group on Asp247. Mutation at position 38 causes a downshift of 3 cm-1 in the v(Fe-Im) stretching mode, suggesting a weakening of the Fe-Im bond strength. By comparing the results obtained with HRP-C* mutants with those previously reported for the corresponding cytochrome c peroxidase (CCP) mutants, it appears that the distal heme pocket architecture is significantly different in the two peroxidases, although the hydrogen-bonding network coupling the distal and the proximal sides of the heme appears to be conserved. Mutations on the distal side dramatically affect the capability of the protein to bind BHA. F41W and R38K mutants do not bind the substrate, whereas the F41V variant shows a 2-fold increase in affinity.(ABSTRACT TRUNCATED AT 400 WORDS)
FMRFaraide-related peptides have been isolated from both invertebrates and vertebrates and exhibit a wide range of biological effects in rats. We show here that in humans 2 FMRFamide-related peptides are encoded by a single gene expressed as a spliced mRNA. The larger predicted peptide (AGEGLNSQFWSLAAPQRFamide) differs from the peptide isolated from bovines (AGEGLSSPFWSLAAPQRFamide) by the substitutions of 2 amino acids. The shorter predicted peptide (NPSF, SQAFLFQPQRFamide) is 3 amino acids longer than the bovine 8 amino-acid NPFF (FLFQPQRFamide) or the human NPFF peptide isolated from serum [5], suggesting that the encoded protein is subject to cleavage by a tripeptidyl peptidase or by a novel processing mechanism. On rat spinal cord, the larger peptide is indistinguishable in activity from the equivalent bovine peptide whereas the smaller extended peptide is inactive.© 1997 Federation of European Biochemical Societies.Key words: NPFF; NPAF; FMRFamide; cDNA; Nociception; Human In bovine brain, 3 different forms of immunoreactive material were detected by HPLC separation and the use of an antibody raised against the molluscan peptide FMRF-NH 2 . Two of these correspond to NPFF and NPAF ; the structure of the third is unknown [12]. Similarly, in the rat hypothalamus, NPFFs have been shown to exist by HPLC and immunoreactivity but the predominant form of peptide is not NPFF [13].The isolation of the human gene described here now allows a definitive description of the human FMRFamide peptides. We show that there is an additional 3 amino acids on the human NPFF precursor that must be cleaved to produced biologically active NPFF. If these amino acids are present on the precursor of the bovine and rat NPFF then this could account for the alternate form detected by HPLC. We also demonstrate that human NPAF is capable of modulating neural activity in the rat spinal cord in a manner indistinguishable from that of bovine NPAF while human N-terminally extended NPFF has no activity.
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