PIF1 DNA helicase from Saccharomyces cerevisiae. Biochemical characterization of the enzyme.

Overexpressed PIF1 DNA helicase was purified from mitochondria to near homogeneity. Its ATPase and unwinding properties were characterized. The enzyme specifically utilizes ATP (or dATP) and MgCl2 (and to a lesser extent MnCl2). ATPase activity requires single-stranded DNA as an effector, duplex DNA being 100-fold less effective. The Keff, defined as the concentration of DNA required to achieve half-maximal ATPase activity, does not depend on single-stranded DNA length. Long duplex DNAs are poorly unwound and, moreover, dilution of the enzyme and its DNA substrate in the assay decreases DNA helicase activity. These data indicate that PIF1 helicase is a distributive enzyme, frequently turning from one DNA molecule to another. When forked substrates are used, unwinding by PIF1 is markedly stimulated. The enzyme has a sedimentation coefficient of 6.5 S, suggesting that it exists as a monomer in solution.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

Suicide notes in adolescence.

Suicide rates for adolescents have shown a substantial increase over the past 30 years, but there is little information regarding the clinical status of adolescents who end their lives. In the adult literature, one avenue to understanding the psychologic condition immediately prior to the self-destructive act has been the study of suicide notes, and the present study constitutes the first systematic investigation of notes left by children or adolescents. Records of death were examined in the Office of the Coroner, City of Montreal, and all suicides between ages 10 and 20 were identified for the years 1978 to 1982. Seventeen individuals who left notes were identified, comprising 10% of the population of suicides. Suicides who left notes did not differ from the total group in age and sex distribution but were more likely to choose shooting as a method. The content of the notes was studied in terms of 11 variables which had proven characteristic of suicide notes in the adult literature, and the results were compared to those reported for adults. In general, our results support a psychoanalytic perspective which understands suicide as resulting from an ambivalent attachment to an object, loss of the object, internalization, and the direction of aggression against the self. Cases appeared to fall into two clusters. Older adolescents were more concrete, left specific instructions, did not address the note, did not give a reason for the act, and tended to choose intoxication as a method.(ABSTRACT TRUNCATED AT 250 WORDS)

An epidemiologic study of adolescent suicide.

This study investigated suicides by persons aged ten to nineteen during the years 1978 to 1982 in the City of Montreal. Records of death in the Office of the Coroner, City of Montreal, were examined and suicides designated according to standard criteria. Age, sex, method of suicide, and month of death were noted. Mean incidence of suicide for the entire sample was 5.92 per 100,000 population. The mean figure for males was 9.52 and for females 2.32, with the incidence greater among males in each of the five years. Incidence of suicide in the fifteen to nineteen year old group was approximately ten times that in the ten to fourteen year old group, a difference which was constant across sex and across the five year period. Among boys, hanging was the most frequent method of suicide, with firearms second, and jumping from a height or in front of a vehicle third. Among girls, drug intoxication was most common, with firearms and jumping ranking second and third respectively. No monthly periodicity was found. The epidemiology of adolescent suicide in Montreal appears to be similar to that reported in other locations.

Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA.

The nuclear gene PIF of Saccharomyces cerevisiae is required for both repair of mitochondrial DNA (mtDNA) and recognition of a recombinogenic signal characterized by a 26-bp palindromic AT sequence in the ery region of mtDNA. This gene has been cloned in yeast by genetic complementation of pif mutants. Its chromosomal disruption does not destroy the genetic function of mitochondria. The nucleotide sequence of the 3.5-kb insert from a complementing plasmid reveals an open reading frame encoding a potential protein of 857 amino acids and Mr = 97,500. An ATP-binding domain is present in the central part of the gene and in the carboxy-terminal region a putative DNA-binding site is present. Its alpha helix-turn-alpha helix motif is found in DNA-binding proteins such as lambda and lactose repressors which recognize symmetric sequences. Significant amino acid homology is observed with yeast RAD3 and E. coli UvrD (helicase II) proteins which are required for excision repair of damaged DNA.