Purification and characterization of yeast topoisomerase I

Yeast topoisomerase I (Mr = 76,000) has been purified to 80% homogeneity using a combination of ion exchange, gel filtration, and DNA-cellulose chromatography. The enzyme was characterized with respect to its ability to relax supercoiled DNA and to catenate nicked circular DNA. Yeast topoisomerase I will remove both positive and negative turns in DNA supercoils in the absence of ATP and magnesium ion. The products of the catenating activity of the enzyme were examined on agarose gels and in the electron microscope. These analyses indicate that yeast topoisomerase I will generate large catenated DNA networks which appear to rearrange to multimeric linear structures upon long incubation time.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Increase in brain 125I-cholecystokinin (CCK) receptor binding following chronic haloperidol treatment, intracisternal 6-hydroxydopamine or ventral tegmental lesions

Specific 125I-CCK receptor binding was significantly increased in brain tissue taken from guinea pig or mouse following chronic (2-3 week) daily administration of haloperidol (2-3 mg/kg/day). Scatchard analysis indicated the increase in CCK binding was due to an increased receptor number (B max) with no change in affinity (Kd). In guinea pigs, the increased CCK binding was observed in the mesolimbic regions and frontal cortex, but not in striatum, hippocampus nor posterior cortex. In mice, however, the increases occurred in both pooled cerebral cortical-hippocampal tissue, and in the remainder of the brain. Enhanced CCK receptor binding was also observed in membranes prepared from whole brain of mice one month following intracisternal injection of 6-hydroxydopamine. Additionally, an increase in CCK binding was observed in mesolimbic regions and frontal cortex, but not striatum or hippocampus, of guinea pigs 3 weeks after an unilateral radiofrequency lesions of the ipsilateral ventral tegmentum. The present studies demonstrate that three different procedures which reduce dopaminergic function in the brain enhance CCK receptor binding. The data provide further support for a functional interrelationship between dopaminergic systems and CCK in some brain regions and raise the possibility that CCK may play a role in the antipsychotic action of neuroleptics.     

Adducts formed between deoxyguanosine and cis-Dichlorodiammineplatinum(II): Separation by means of cation-exchange chromatography

The interaction between deoxyguanosine (dG) and cis-dichlorodiammineplatinum(II) (cis-Pt) leads to the 2:1 and the 1:1 dG-Pt adducts. These adducts were separated on an Aminex A6 cationexchange column by use ot 0.01 M K₂CO₃ (pH 11) as an eluent. The stoichiometry of the adducts was determined from the ^195mPt radioactivity and from the absorbance of the guanine chromophore at 280 nm. Time-course studies show that dG reacts initially with cis-Pt to form the 1:1 adduct, which then interacts with a second molecule of dG to form the 2:1 adduct. Acid hydrolysis (100°C in 88% formic acid for 5–15 min) of the 1:1 and 2:1 adducts results in their conversion to two new products, which elute differently from the column but which still contain Pt bound in the same stoichiometric ratio to dG as in the nonhydrolyzed adducts. The hydrolyzed adducts show a negative diphenylamine reaction indicative ot cleavage of the glycosidic bond. It is concluded that mild acid hydrolysis converts the 1:1 and 2:1 dG-Pt adducts into the corresponding guanine-Pt adducts, which are chromatographically distinguishable. This acid hydrolysis-high pressure liquid chromatography (HPLC) procedure has application to the identification of the Pt adducts formed in DNA.     

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.     

Genetic and biochemical distinction among Chinese hamster cell emtA, emtB, and emtC mutants.

Genetic and biochemical experiments have enabled us to more clearly distinguish three genetic loci, emtA, emtB, and emtC, all of which can be altered to give rise to resistance to the protein synthesis inhibitor, emetine, in cultured Chinese hamster cells. Genetic experiments have demonstrated that, unlike the emtB locus, neither the emtA locus nor the emtC locus is linked to chromosome 2 in Chinese hamster cells, clearly distinguishing the latter two genes from emtB. emtA mutants can also be distinguished, biochemically, from emtB and emtC mutants based upon different degrees of cross-resistance to another inhibitor of protein synthesis, cryptopleurine. Two-dimensional gel electrophoretic analysis of ribosomal proteins failed to detect any electrophoretic alterations in ribosomal proteins from emtA or emtC mutants that could be correlated with emetine resistance. However, a distinct electrophoretic alteration in ribosomal protein S14 was observed in an emtB mutant. In addition, the parental Chinese hamster peritoneal cell line of an emtC mutant, and the emtC mutant itself, are apparently heterozygous for an electrophoretic alteration in ribosomal protein L9.     

Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.