Identification of a DinB/UmuC homolog in the archeon Sulfolobus solfataricus.

To date, eight closely related homologs of the Escherichia coli UmuC protein have been identified. All of these homologs appear to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. Recently, a distantly related UmuC-homolog, DinB, has also been identified in E. coli. Using the polymerase chain reaction together with degenerate primers designed against conserved regions found in UmuC-like proteins, we have identified a new member of the UmuC-superfamily in the archeon Sulfolobus solfataricus. This new homolog shows high sequence similarity to DinB and a lower level of similarity to UmuC. As a consequence, we have called this new gene dbh (dinB homolog). Analysis of approximately 2.7 kb DNA encompassing the dbh region revealed several open reading frames (orfs). One, encoding a putative ribokinase, was located immediately upstream of dbh. This orf overlaps the dbh gene by 4 bp suggesting that both proteins might be coordinately expressed. Further upstream of the ribokinase-dbh locus was another orf encoding a potential ATPase homologous to two uncharacterized S. cerevisiae proteins (YD9346.02c and SC38KCXVI_20) and another E. coli DNA repair protein, RuvB. While this is the first report of a UmuC-like homolog in an archeon, we detected additional homologs using protein sequence comparisons in Gram-positive bacteria, cyanobacteria, and among potential human EST products, indicating that UmuC-related proteins comprise a ubiquitous superfamily of proteins probably involved in DNA repair and mutagenesis.

A novel Tn10 tetracycline regulon system controlling expression of the bacteriophage T3 gene encoding S-adenosyl-L-methionine hydrolase.

To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adenosyl-L-methionine (AdoMet). The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine and 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo. The amh gene was placed downstream from the inducible tetA promoter of the Tn10 tetracycline regulon substituting for most of the tetA gene. Unlike in the original isolates [Hughes et al., J. Bacteriol. 169 (1987) 3625-2632], this promoter allows controlled expression. These constructs are stable and can be induced in a dose-dependent manner. The system is maximally induced 2-3 h after addition of the inducer, autoclaved chlortetracycline (cTc). DNA methylation in vivo was assessed in this model system by BamHI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M.BamHII methyltransferase encoding gene. The induction of amh decreased the intracellular pool of AdoMet which M.BamHII requires as a cofactor. Under these conditions, there is a decrease in DNA methylation. The unmethylated DNA is assayed by BamHI cleavage. This system will be useful for studying transcription, DNA replication, gene repair and other cellular phenomena affected by methylation.

Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide.

To characterize the mechanism of membrane attachment of dopamine beta-hydroxylase, an expression system producing the processed form of this enzyme has been developed. We have replaced the endogenous signal peptide of bovine dopamine beta-hydroxylase with a heterologous signal peptide which is efficiently recognized and cleaved in Drosophila Schneider 2 cells. A cDNA encoding this chimeric recombinant bovine enzyme has been stably transfected into Schneider 2 cells. The inducible expression of active dopamine beta-hydroxylase in these cells has been verified by Western blotting and enzyme activity assays. N-terminal sequence analysis of purified recombinant enzyme demonstrates complete removal of the signal peptide. Subcellular analysis shows that the recombinant enzyme exists as both a soluble and a membrane-bound form in these cells. These data demonstrate that the endogenous signal peptide is not required for the formation of the membranous dopamine beta-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence.

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens.

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E.coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E.coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis.

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.

Perinatal addiction: outcome and management.

The care of the pregnant drug-dependent woman and her newborn infant has become a major and controversial health problem requiring specific approaches to this high-risk mother and neonate. A comprehensive approach to the care of 278 pregnant drug-dependent women and their infants at the Philadelphia General Hospital has significantly reduced maternal and infant morbidity heretofore associated with pregnancies complicated by opiate addiction. Most significantly, the incidence of low birth weight has been reduced to below 20 per cent, and a decrease in severe withdrawal in infants born to mothers in the comprehensive care program has occurred. We propose that application of this approach to women whose pregnancies are complicated by drug dependency is a significant factor in successful management.

The effects of maternal drug dependence on neonatal mortality.

Addiction in pregnancy has become an important health problem owing to the tendency of drug-dependent women to neglect general health care and to avoid seeking prenatal care. In addition, continued heroin administration during pregnancy carries additional risks for the maternal-fetal unit. Thus, there is an increased incidence of obstetrical and medical complications in these mothers, resulting in high incidences of prematurity, low birth weight and mortality in their infants. Therefore, there is a high neonatal mortality rate due to clinical conditions most commonly seen among premature infants. Data from three groups of drug-dependent women and their infants and one control group demonstrate that the high mortality rate, as well as the incidence of low birth weight, can be reduced to a rate similar to the control group in infants of mothers who receive comprehensive services that include prenatal care in conjunction with methadone maintenance.

Intravascular volume determinations and fetal outcome in hypertensive diseases of pregnancy.

With the Evans blue technique, 178 plasma volume, blood volume, and red cell volume determinations were studied in 51 hypertensive and 35 normotensive gravidas. Hypertensive pregnancies were classified according to the American Committee on Maternal Welfare schema. A large range and overlap in plasma and blood volume determinations was found, but mean values in patients with hypertensive disease were significantly depressed when compared with normotensive gravidas. A decrease in red cell mass was observed only in the severe hypertensive group of women. In general, those hypertensive patients with plasma and blood volume determinations approaching those of the normotensive control patients were associated with a favorable maternal and fetal outcome. Extreme hypovolemia occurred in most pregnancies associated with severe hypertensive disease and evidence of uteroplacental insufficiency.

Assessment and treatment of abstinence in the infant of the drug-dependent mother.

To accomplish the assessment and treatment of abstinence in the infant of the drug dependent mother, a scoring system has been devised for use as a clinical and research tool. The score monitors the passively addicted infant in a more comprehensive and precise way than has previously been described, and permits us to be more objective in our clinical estimates of the abstinence syndrome. Further, this scoring system has been used by relating it to the dosage schedule of phenobarbital or paregoric as part of an ongoing research project designed to test the comparative usefulness of recommended treatments for neonates with abstinence symptoms. It has been found useful also in following the progression or diminution of symptomatology before, during and after drug therapy. Moreover, the scoring system provides a basis for developing uniform criteria for the assessment and treatment of the neonate of the addicted mother.

Neonatal abstinence syndrome: assessment and management.

A scoring system for the neonatal abstinence syndrome has been devised and implemented as both a clinical and investigative tool. The score monitors the passively addicted infant in a more comprehensive and objective fashion, and facilitates a more precise evaluation of the clinical status of the infant undergoing withdrawal. In addition, the scoring system has been applied in research designed to test the comparative usefulness of various pharmacologic agents currently recommended for the neonatal abstinence syndrome, and has been found useful in following the progression and diminution of withdrawal symptomatology before, during, and after therapy. Furthermore, the scoring system provides a basis ofr developing uniform criteria for the assessment and treatment of the neonate born to the addicted mother.