The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.     

Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates

We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, SalI, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactams, chloramphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occurred in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype.     

Human chromosomal assignments for 14 argininosuccinate synthetase pseudogenes: cloned DNAs as reagents for cytogenetic analysis

There are multiple, processed, dispersed pseudogenes for human argininosuccinate synthetase. Chinese hamster X human somatic cell hybrids were used to map DNA fragment groups corresponding to the single expressed gene and 14 pseudogene loci. Each chromosomal assignment was confirmed using hybrids containing very few human chromosomes and/or by demonstrating monosomic or trisomic dosage in human cell lines with chromosomal abnormalities. Pseudogenes were mapped to chromosomes 2cen-p25, 3q12-qter, 4q21-qter, 5 (two loci), 6, 7, 9p13-q11, 9q11-q22, 11q, 12, Xp22-pter, Xq22-q26, and Ycen-q11. DNA fragments from the expressed gene were mapped to 9q34-qter in agreement with the previous assignment for enzyme activity. A high-frequency restriction fragment length polymorphism mapped to 9q11-q22. The analyses emphasized the feasibility of using chromosomally abnormal human cell lines for confirmation and regionalization of gene-mapping assignments made using somatic-cell hybrids. Conversely, cloned DNA probes, once mapped and characterized, can be very valuable for determining the chromosomal composition of interspecies hybrids and the dosage of loci in human cells. The argininosuccinate synthetase cDNA is a convenient reagent for dosage analysis of 15 human loci on 11 different chromosomes. Improved reagents could be designed that would simplify Southern blot patterns by eliminating overlapping DNA fragments and providing a single DNA fragment for each locus.     

The effects of antioxidants on the metabolism and mutagenicity of benzo[a]pyrene in vitro.

Antioxidants inhibit the rat liver microsomal mixed-function-oxidase-catalysed hydroxylation of benzo[a]pyrene. These antioxidants also decrease the formation of mutagenic products from benzo[a]pyrene as judged by the Ames bacterial-mutagenicity assay [B.N. Ames, J. McCann & E. Yamazaki (1975) Mutat. Res. 31, 347-364]. It is suggested that antioxidants exert their protective effect against cancer by inhibiting the formation of carcinogenic metabolites.     

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins.

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.     

Complementation Mapping of Skeletal and Central Nervous System Abnormalities in Mice of the Piebald Deletion Complex

The s(15DttMb), s(36Pub), s(1Acrg) and s(24Pub) piebald deletion alleles belong to a set of overlapping deficiencies on the distal portion of chromosome 14. Molecular analysis was used to define the extent of the deletions. Mice homozygous for the smallest deletion, s(15DttMb), die shortly after delivery and display alterations in the central nervous system, including hydrocephalus and a dorsally restricted malformation of the spinal cord. These mice also display homeotic transformations of vertebrae in the midthoracic and lumbar regions. Homozygous s(27Pub) mice contain a point mutation in the piebald gene, survive to weaning, and display no central nervous system or skeletal defects, arguing that the s(15DttMb) phenotype results from the loss of genes in addition to piebald. A larger deletion, s(36Pub), exhibits additional cartilage malformations and defects in the anterior axial and cranial skeleton. The skeletal defects in both s(15DttMb) and s(36Pub) mice resemble transformations associated with the targeted disruption of Hox genes and genes encoding the retinoic acid receptors, which play a role in the specification of segmental identity along the anteroposterior axis. Complementation analysis of the s(15DttMb) and s(36Pub) phenotypes, using two additional deletions, localized the gene(s) associated with each phenotype to a defined chromosomal region.     

LIPID COMPOSITION OF OPTIC NERVE MYELIN

Abstract— Myelin was isolated from bovine optic nerves by differential ultracentrifugation and its lipid composition was analysed. Optic nerve myelin contained 76·3 per cent lipid. The major lipids were cholesterol, ethanolamine glycerophosphatides (EGP) and cerebroside. Serine glycerophosphatides (SGP), sphingomyelin and cerebroside sulphate were present in smaller proportions. EGP and SGP contained 34·6 and 0·5 per cent aldehydes. The major fatty aldehydes were palmitaldehyde, stearaldehyde and octadecenaldehyde. The fatty acids of EGP, SGP and choline glycerophosphatides (CGP) were chiefly 16:0, 18:0 and 18:1, with small proportions of 20 and 22 carbon polyunsaturates. The sphingolipids contained predominantly saturated and monounsaturated fatty acids of chain lengths of 20–26 carbon atoms. Optic nerve myelin and white matter myelin resembled one another closely in overall lipid composition and in the fatty acid compositions of their constituent lipids. Optic nerve myelin and white matter myelin are chemically similar membranes, but both of these differ in their lipid composition from spinal root myelin.     

Polyol-pathway enzymes of human brain. Partial purification and properties of aldose reductase and hexonate dehydrogenase.

Aldose reductase and hexonate dehydrogenase were isolated from human brain and partially purified. The two enzymes exhibited distinctive substrate-specificity profiles with a variety of aldoses,and aliphatic and aromatic aldehydes. Aldose reductase exhibited a high affinity for DL-glyceraldehyde (Km of 62 microM) and a low affinity (Km of 90 mM) for glucose, the physiological substrate of the polyol pathway. Hexonate dehydrogenase exhibited a relatively low affinity for D-glucuronate (Km of 4.6 mM) and a very low affinity for glucose (Km of 390 mM). Both enzymes exhibited a high specificity for NADPH, and both were inhibited competitively by NADP+. Hexonate dehydrogenase was inhibited by iodoacetate, iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate. Preincubation with 2-mercaptoethanol resulted in activation. Both enzymes were inhibited by a number of barbiturates (barbital, phenobarbital and pentobarbital) and by the central-nervous-system drugs diphenylhydantoin and ethosuccinimide. The substrate specificity and pattern of inhibition suggest that the two enzymes isolated correspond to two of four previously reported aldehyde reductases isolated from human brain.