Yeasts

Science, 1993 Dec 17, 262(5141), 1889 - 92
Inhibition of transcriptional regulator Yin-Yang-1 by association with c-Myc; Shrivastava A et al.; Yin-Yang-1 (YY1) regulates the transcription of many genes, including the oncogenes c-fos and c-myc . Depending on the context, YY1 acts as a transcriptional repressor, a transcriptional activator, or a transcriptional initiator . The yeast two-hybrid system was used to screen a human complementary DNA (cDNA) library for proteins that associate with YY1, and a c-myc cDNA was isolated . Affinity chromatography confirmed that YY1 associates with c-Myc but not with Max . In cotransfections, c-Myc inhibits both the repressor and the activator functions of YY1, which suggests that one way c-Myc acts is by modulating the activity of YY1.

Nature, 1993 Dec 16, 366(6456), 698 - 701
A first-generation physical map of the human genome; Cohen D et al.; Sets of ordered overlapping cloned genomic DNA fragments that span each of the human chromosomes are urgently needed for identification of human disease genes . Such a physical map also provides unique material to study the structure and function of the genome . We have therefore exhaustively analysed the CEPH yeast artificial chromosome (YAC) library, which contains 33,000 clones, whose insert size was individually determined . These YACs have an average length of 0.9 megabases and cover the equivalent of 10 haploid genomes . Several mapping techniques were combined to provide multiple sources of structural information for most of these clones . Finally, the library was screened with more than 2,000 genetic markers quasiuniformly distributed over 90% of the genome . These results should allow the scientific community to construct detailed maps of all human chromosomes . Moreover, we propose a data analysis strategy that produces a first-generation integrated map covering most of the human genome.

Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 639 - 45
Expression of APP in brains of transgenic mice containing the entire human APP gene; Buxbaum JD et al.; A major component of amyloid deposits found in Alzheimer disease and Down syndrome is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP) . Recent evidence indicates that increases in APP expression and/or beta/A4 peptide accumulation may underlie the amyloidosis characteristic of these diseases . In the present study, transgenic mice carrying the entire human APP gene were studied for expression of human APP . Significant expression of human APP protein was observed in these animals, and this expression paralleled the expression of endogenous APP . These results, which represent a first demonstration of significant human APP expression in transgenic animals, support the use of such animals to study human APP expression and processing in vivo and possibly as models for the amyloidosis associated with Alzheimer disease.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11870 - 4
Genes in one megabase of the HLA class I region; Wei H et al.; To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex . These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F . In addition to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by us and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region . We also identified a long repetitive DNA element in the region between HLA-B and HLA-E . Homologues of this element were located at several sites in the human genome outside of the HLA complex . The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions . Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.

EMBO J, 1993 Dec 15, 12(13), 5181 - 9
Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo; Cortes JJ et al.; We have analyzed base pairing interactions between the U5 snRNA and 5' exon sequences during pre-mRNA splicing in a mammalian in vivo system . We constructed synthetic U5 genes with mutations that alter four bases (C3, U4, U5 and U6) within the invariant 9 nt U5 sequence GCCUUUUAC; transient transfection of HeLa cells with these U5 sequences cloned into a U1 expression vector yielded high levels of the mutant snRNAs . To test their function, we cotransfected a rabbit beta-globin gene containing one of two mutations (G1-->A or T2-->A) in the essential GT dinucleotide at the 5' end of the second intron . Certain U5 loop mutants activated novel 5' splice sites only in mutant rabbit beta-globin transcripts . One novel site surprisingly resides in the first exon; its use is invariably coupled to utilization of a particular cryptic 5' splice site in the second exon . All of the newly activated cryptic 5' splice sites exhibit complementarity with the mutant U5 loop in the exon 1-5 nt upstream of the cryptic site, extending previous results in yeast . However, the register of the potential pairing is not identical at the various novel cryptic 5' splice sites, indicating that the interaction between the U5 loop and the 5' exon may be more flexible than previously believed.

Cancer Res, 1993 Dec 15, 53(24), 5934 - 9
Expression of a constitutively active estrogen receptor variant in the estrogen receptor-negative BT-20 human breast cancer cell line; Castles CG et al.; Estrogen receptor (ER) expression by breast tumors is an important predictor of disease-free survival in breast cancer patients and, more importantly, is a strong predictor of response to endocrine therapy . Variant forms of the ER may play an important role in the loss of hormone responsiveness and the progression to hormone independence . We have examined a panel of human breast tumor cell lines, both ER-positive and ER-negative, and have identified an ER mRNA variant containing a deletion of exon 5 in the ER-negative BT-20 and ER-positive MCF-7 cell lines . This exon 5 deletion variant has been previously reported to be overexpressed in ER-negative/progesterone receptor-positive breast tumors . Using RNase protection analysis, we have found that the predominant ER transcript in the BT-20 cells is the exon 5 deletion variant, while the principal transcript in MCF-7 cells is the wild-type ER mRNA . The variant ER transcript is translated into a truncated receptor protein of approximately M(r) 42,000 when expressed in yeast and, more important, in breast tumor cells . This is the first demonstration of an exon 5 deletion variant ER protein . Functional analysis has shown that this variant ER possesses constitutive transcriptional regulatory activity with respect to an estrogen-regulated reporter gene construct in a yeast expression system . The presence of this ER variant in breast tumor cell lines, as well as breast tumor biopsies and uterine tissue, suggests that it is a naturally occurring variant that may arise by alternative splicing, and whose overexpression may be involved in the progression of breast tumors to a hormone-independent state.

Biochim Biophys Acta, 1993 Dec 8, 1203(2), 215 - 23
Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11 beta-hydroxylation in Curvularia lunata; Suzuki K et al.; Addition of 11-deoxycortisol to the culture medium of Curvularia lunata induced the increase of cytochrome P-450 content and steroid 11 beta-hydroxylase activity . The enzyme in cell-free extract produces cortisol from 11-deoxycortisol in the presence of NADPH and O2 . The enzyme was partially stabilized by glycerol, 11-deoxycortisol, GSH and PMSF . The hydroxylation activity was strongly inhibited by carbon monooxide and sulfhydryl reagents . Cytochrome P-450 located on the microsomal fraction was solubilized with Triton X-100 and sodium cholate and purified to apparent homogeneity by column chromatography . The purified cytochrome P-450 (P-450lun) has a molecular mass of 60 kDa and exhibits the absorption maximum at 392 nm in the spectrum of oxidized form in the presence of 11-deoxycortisol . The reduced CO difference spectrum has a maximal peak at 448 nm . 11 beta-Hydroxylation of 11-deoxycortisol was reconstituted by cytochrome P-450lun, C . lunata NADPH-cytochrome P-450 reductase and DLPC in the presence of NADPH and O2 with a turnover number of 207 nmol/min per nmol of cytochrome P-450 . The reductase and DLPC could be partially replaced with the enzyme purified from yeast or pig testis microsome and lipids purified from C . lunata, respectively . P-450lun catalyzes bifunctionally 11 beta- and 14 alpha-hydroxylations of 11-deoxycortisol . Deoxycorticosterone, progesterone, androstenedione and testosterone are hydroxylated in the similar manner.

Biochemistry, 1993 Dec 7, 32(48), 13138 - 45
Site-specific cleavage at a DNA bulge by neocarzinostatin chromophore via a novel mechanism; Kappen LS et al.; The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) oxidatively cleaves single-stranded or duplex DNA site-specifically in the absence of activating thiol provided that the DNA contains a bulged structure . Point mutations, deletions, and insertions in the DNA analogue and its complement of the 3'-terminus of yeast tRNA(Phe) show that for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must generate a hairpin structure with an apical loop and at least a two-base-pair stem hinged to a region of duplex structure via a bulge containing a target nucleotide at its 3' side . The size of the loop is not critical so long as it contains at least three nucleotides; the bulge requires a minimum of two nucleotides but must have fewer than five . With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom . Maintenance of the bulged structure requires stable duplex regions on each side of the bulge . A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cleavage in the thiol-independent reaction . Drugs such as netropsin, which sequester the DNA into nonbulge containing structures inhibit the reaction . In the absence of O2 strand cleavage is blocked and quantitatively replaced by a presumed drug-DNA covalent adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Dec 5, 234(3), 661 - 79
Affinity and specificity of serine endopeptidase-protein inhibitor interactions . Empirical free energy calculations based on X-ray crystallographic structures; Krystek S et al.; An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase . Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes . The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH . In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0) . The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops . The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation . In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes . In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal) . In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7) . Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)

Semin Dermatol, 1993 Dec, 12(4), 276 - 9
Pityriasis versicolor; Faergemann J; The lipophilic yeast Pityrosporum ovale is both a member of the normal human cutaneous flora in adults and the etiological agent of pityriasis versicolor . Pityriasis versicolor develops under the influence of predisposing factors . The presence of these factors are also the reason for the high rate of recurrence seen in pityriasis versicolor and for its chronicity . There are numerous ways of treating pityriasis versicolor topically and systemically . Propylene glycol 50% in water is effective and cheap, but the imidazoles and the older antidandruff shampoos as well as two new antifungals: ciclopiroxolamine and terbinafine are also effective topically . However, short-term oral treatment with ketoconazole, itraconazole or fluconazole are very effective and the risk for side effects minimized with short treatment regiments . The patient compliance is also higher with oral treatment . The recurrence rate is very high, and to avoid this a prophylactic treatment schedule (eg, ketoconazole) one 200 mg tablet on three consecutive days every month or a single dose of 400 mg every month are effective.

Genomics, 1993 Dec, 18(3), 553 - 8
Construction and characterization of a YAC library with a low frequency of chimeric clones from flow-sorted human chromosome 9; McCormick MK et al.; Human chromosome 9 DNA, flow-sorted from somatic cell hybrid PK-87-9, has been used to construct two complete digest YAC libraries . The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is approximately 95% . The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be approximately 4% . These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.

Genomics, 1993 Dec, 18(3), 546 - 52
A complete YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13) and refined localization of the SNRPN gene; Mutirangura A et al.; Since a previous report of a partial YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13), a complete contig spanning approximately 3.5 Mb has been developed . YACs were isolated from two human genomic libraries by PCR and hybridization screening methods . Twenty-three sequence-tagged sites (STSs) were mapped within the contig, a density of approximately 1 per 200 kb . Overlaps between YAC clones were identified by Alu-PCR dot-blot analysis and confirmed by STS mapping or hybridization with ends of YAC inserts . The gene encoding small nuclear ribonucleoprotein-associated peptide N (SNRPN), recently identified as a candidate gene for Prader-Willi syndrome, was localized within this contig between markers PW71 and TD3-21 . Loci mapped within and immediately flanking the Prader-Willi/Angelman chromosome region contig are ordered as follows: cen-IR39-ML34-IR4-3R-TD189-1-PW71-SNRPN -TD3-21- LS6-1-GABRB3,D15S97-GABRA5-IR10-1-CMW1+ ++-tel . This YAC contig will be a useful resource for more detailed physical mapping of the region, for generation of new DNA markers, and for mapping or cloning candidate genes for the Prader-Willi and Angelman syndromes.

Genomics, 1993 Dec, 18(3), 486 - 95
A somatic cell hybrid map of human chromosome 13; Washington SS et al.; We have constructed a chromosome 13 somatic cell hybrid map using seven cell lines: PGMEA6, a hybrid containing the entire chromosome 13, and six hybrids containing various deletions of chromosome 13 (BARF7, PPF22, KBF11, KSF39, CF25, and CF27) . We have mapped 80 markers that define 10 regions of chromosome 13 with respect to 10 breakpoints in the mapping panel; these regions range in size from 4 to 24 Mb, with an average size of 8 Mb . The 80 markers sublocalized on our mapping panel include 10 Alu-PCR clones, 6 of which were converted to sequence-tagged sites; 40 (CA)n repeat-containing clones, 27 of which are microsatellite PCR markers; 8 (AAAG)n repeat-containing PCR markers, 1 two-allele PCR marker, 4 genes or expressed sequences, and 17 anonymous DNA probes . This low-resolution physical map can be used as a backbone map for more refined physical mapping using radiation hybrids or yeast artificial chromosomes.

Yakugaku Zasshi, 1993 Dec, 113(12), 829 - 46
{The characterization of human cdc2 kinase and CDK2}; Yasuda H et al.; p34cdc2 kinase plays a key role in the initiation of mitosis . The activity of this kinase requires the binding of a protein, named cyclin, to it . The kinase forms a heterodimer with cyclin . Cyclin A or B is the counterpart of this complex . The differences in the activity between cyclin A/cdc2 kinase and cyclin B/cdc2 kinase have not been cleared . In recent years, the other cdc2-like kinases were identified . One of them was CDK2 (cyclin dependent kinase 2) . CDK2 could rescue the defect of the budding yeast CDC28 mutation, which arrested the cells at a point named START, in G1 phase . Then, CDK2 was thought to be worked at G1 through S phase in a cell cycle, but the details on the role of this kinase has not been cleared so far . In this study, we separated the human cyclin A/cdc2 kinase, cyclin B/cdc2 kinase and CDK2, each other by use of column chromatography, and characterized the each kinase . These kinases had the same substrate specificities when the synthesized peptides were tested . They phosphorylated the threonine residue in the sequence -Thr-Pro-Lys-Lys-Ala- but hardly phosphorylated threonine residue the sequence -Thr-Pro-Lys-Ala-Lys- . They had some differences in the substrate-preference when the native proteins were tested . In a cell cycle of human cells, the activity of cdc2 kinase increased at G2/M phase and the activity of CDK2 was high from S through M phase . These data suggested that cdc2 kinase works at the transition from G2 to M phase and that CDK2 works from G1 through G2/M phase . They could phosphorylate different protein-substrates having the common phosphorylated sequence -Thr-Pro-X-Lys-.

Nat Genet, 1993 Dec, 5(4), 338 - 43
Mapping, cloning and genetic characterization of the region containing the Wilson disease gene; Petrukhin K et al.; Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3 . In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region . Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval . A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene . Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

Biotechnol Appl Biochem, 1993 Dec, 18 ( Pt 3), 275 - 83
Effects of 1,5-anhydro-D-fructose on selected glucose-metabolizing enzymes; Taguchi T et al.; It was verified, by n.m.r . and fast-atom-bombardment-m.s . studies, that the C-2 position of 1,5-anhydro-D-fructose, which was prepared by the reaction of immobilized glucose 2-oxidase from Coriolus versicolor (with 1,5-anhydro-D-glucitol), is hydrated to the acetal form in water . The effects of 1,5-anhydro-D-fructose on several glucose-metabolizing enzymes were compared with those of 1,5-anhydro-D-glucitol . Glucose 1-oxidase from Aspergillus niger was inhibited by 1,5-anhydro-D-fructose (Ki 6.6 mM) more effectively than 1,5-anhydro-D-glucitol (Ki 82.5 mM) . Yeast and rat brain hexokinases phosphorylated 1,5-anhydro-D-fructose (Km,yeast 2.3 mM: Km,rat 0.79 mM) and 1,5-anhydro-D-glucitol (Km,yeast 3.9 mM; Km,rat 0.83 mM) . The phosphorylated forms of these compounds inhibited D-glucose phosphorylation by yeast hexokinase (Ki of phosphorylated 1,5-anhydro-D-fructose 0.11 mM; Ki of phosphorylated 1,5-anhydro-D-glucitol 0.38 mM) and rat brain hexokinase (Ki of phosphorylated 1,5-anhydro-D-fructose 0.07 mM; Ki of phosphorylated 1,5-anhydro-D-glucitol 0.04 mM) . Glucokinase phosphorylated neither 1,5-anhydro-D-fructose nor 1,5-anhydro-D-glucitol, and the phosphorylation of D-glucose by glucokinase was inhibited by them . Mutarotase was slightly inhibited by 1,5-anhydro-D-fructose, as well as by 1,5-anhydro-D-glucitol.

Scand J Dent Res, 1993 Dec, 101(6), 386 - 90
Miconazole chewing gum for treatment of chronic oral candidosis; Rindum JL et al.; A chewing gum containing the antifungal drug miconazole may be convenient for topical treatment of oral candidosis . Therefore a trial was performed to examine the effect and tolerance of miconazole chewing gum in comparison with miconazole gel in the treatment of oral candidosis . The study group consisted of 32 patients with oral candidosis harboring yeasts, predominantly Candida spp . Half of the patients chewed one piece of chewing gum (dose: 3.6 mg of miconazole) four times daily; the other half dispersed a 2% gel (dose: 50 mg of miconazole) in the oral cavity four times daily . After 6 wk of treatment, there was no clinical evidence of yeast infection in either of the two groups . No significant differences between the two groups were found in clinical, mycologic, and cytologic investigations conducted after 3 and 6 wk of treatment or at the follow-up examination 4 wk after termination of the treatment . The results indicate that miconazole released from chewing gum is as effective as miconazole gel . The chewing gum reduced the dosage of miconazole for treatment of oral candidosis, and the patients approved the chewing gum as a pleasant medicament.

Otolaryngol Clin North Am, 1993 Dec, 26(6), 919 - 40
Basic mycology underscoring medically important fungi; Bottone EJ et al.; This article details the basic mycologic features of yeast and mold-like fungi causing infections in humans . Concordant with the mycologic attributes delineating species identification, the pathogenic potential of mycotic agents is discussed with particular reference to intrinsic fungal virulence factors (e.g., exoenzymes) and host factors (e.g., neutrophil function, underlying disease) predisposing to colonization and infection.

Cancer Genet Cytogenet, 1993 Dec, 71(2), 173 - 5
Translocation of CD3D gene in an acute myeloid leukemia (M5) with t(11;17)(q23;21); Pereira MS et al.; Cytogenetic studies in patients with acute leukemia showed structural abnormalities on chromosome 11 at band q23 in five cases . Four of these had acute lymphoblastic leukemia (ALL) associated with t(4;11)(q21;q23) and one case had acute nonlymphoblastic leukemia (ANLL) (M5) associated with t(11;17)(q23;q21) . We examined the CD3D and c-ets-1 genes in the t(11;17)(q23;q21) patient to ascertain any association between them and the chromosome change . In situ hybridization results showed that unlike in other studied cases with rearrangements of 11q23, the CD3D gene in the t(11;17)(q23;21) is transposed to the der(17) chromosome, providing evidence for a different breakpoint in the 11q23 region.

Plant J, 1993 Dec, 4(6), 993 - 1002
Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana; Kwart M et al.; A general amino acid permease cDNA (AAP2) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake . Direct transport measurements in yeast show that the protein mediates uptake of L-{14C}-citrulline and L-{14C}-proline . Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e . glutamine, glutamate and asparagine . Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain . The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins . Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids . Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen . AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling . Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.

Mol Gen Genet, 1993 Dec, 241(5-6), 586 - 94
An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit; Shirley BW et al.; A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana . This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote . However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential . Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals . Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis . A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.

Hum Genet, 1993 Dec, 92(6), 605 - 14
von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis; Yao M et al.; Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder . We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL . The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19-63' . The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene . The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.

Hum Genet, 1993 Dec, 92(6), 527 - 32
A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones; Popp S et al.; The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems . In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with "whole chromosome painting" probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones . To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated . A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6 . For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23 . These experiments demonstrated a trisomy 6pter-->6p22 and a monosomy 8pter-->8p23 in the patient . The present limitations for a broad application of this strategy and its possible improvements are discussed.

Plant Mol Biol, 1993 Dec, 23(5), 981 - 94
The GAPDH gene system of the red alga Chondrus crispus: promoter structures, intron/exon organization, genomic complexity and differential expression of genes; Liaud MF et al.; Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi . Here we characterize the genomic sequences of genes GapC and GapA of C . crispus with respect to promotor structures, intron/exon organization, genomic complexity, G + C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively . To our knowledge this is the first report on nuclear protein genes of red algae . The GapC gene is G + C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome . The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5' end . This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants . It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of -61.3 kJ . CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated . Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles . Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.

Am J Med, 1993 Dec, 95(6), 584 - 8
Immunogenicity of two recombinant hepatitis B vaccines in older individuals; Treadwell TL et al.; PURPOSE: Currently available hepatitis B vaccines are recombinant, yeast-derived preparations given in 10-micrograms or 20-micrograms doses . The optimum dose remains controversial . We sought to assess the relative immunogenicity of two hepatitis B vaccines, given in different doses, in older individuals . PATIENTS AND METHODS: In a multicenter, double-blind, randomized clinical trial, a total of 460 healthy subjects between 39 and 70 years of age were screened and immunized with either Engerix-B 20 micrograms or Recombivax HB 10 micrograms in standard, intramuscular, 3-dose regimens . Of these, 397 subjects were eligible to continue vaccination . Immunogenicity was measured by determination of antibody to hepatitis B surface antigen (anti-HBs) . Seroconversion and seroprotection rates, and geometric mean titers of anti-HBs were calculated at 1, 3, 6, and 8 months after the initial dose of vaccine . RESULTS: Seroprotection rates for subjects receiving the 20-micrograms dose of vaccine were slightly, but not significantly, greater than for subjects receiving the 10-micrograms dose, at each time point . However, at 3 months, males receiving the higher dose had significantly higher seroprotection rates than males receiving the lower dose: 63% versus 37% (p < 0.001) . At 8 months, geometric mean titers for the group receiving Engerix-B 20 micrograms were significantly greater than that for the group receiving Recombivax HB 10 micrograms: 840 mIU/mL versus 340 mIU/mL (p = 0.001) . CONCLUSIONS: Immunization with the 20-micrograms dose of recombinant hepatitis B virus vaccine appeared to result in more rapid development of seroprotective anti-HBs titers in older men and in higher titers of anti-HBs at the completion of vaccination when compared to the 10-micrograms dose . The latter data suggest that the 20-micrograms dose may result in a longer duration of seroprotective anti-HBs titers.

J Cell Physiol, 1993 Dec, 157(3), 509 - 18
Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: determination of the rate-limiting step; Whitesell RR et al.; The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose . To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes . The method is based on the use of intracellular {2-3H}glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of {2-3H}glucose efflux and of 3H2O released by phosphorylation . The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphorylation activities can be regulated by expression of mRNA or injection of foreign protein . Oocytes microinjected with {2-3H}glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 microM and a Vmax of 0.1 nmol/min/oocyte . Co-injection of yeast hexokinase increased glucose phosphorylation by five-fold . Expression of human glucose transporter (GLUT1) mRNA resulted in a 25-30-fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes . The kinetics of transport and phosphorylation of {2-3H}glucose were analyzed by a multiple curve-fitting program that provided estimates of kinetic coefficients for both processes from a single time course . The analysis showed that expression of GLUT1 shifted the rate-limiting step in glucose utilization from transport to phosphorylation . A similar shift occurred at a three-fold lower extracellular concentration of 2-deoxyglucose . In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step . The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models.

Genes Dev, 1993 Dec, 7(12A), 2378 - 91
Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins; Hannon GJ et al.; A two-hybrid protein interaction screen was used to isolate cDNAs encoding human proteins that can interact with human CDK2 in yeast . A new member of the retinoblastoma susceptibility gene family, Rbr-2 (Rb-related), was obtained . The sequence of the Rbr-2 protein shares approximately 50% identify with p107 and homology to Rb within the pocket domain . Several lines of evidence indicate that Rbr-2 is the adenovirus E1A-associated p130 . Like Rb and p107, p130Rbr-2 can bind to viral oncoproteins, SV40 large T antigen, and adenovirus E1A through its pocket domain . Although p130Rbr-2 does not bind to CDK2 in vitro, it can interact with cyclins, with a clear preference for D-type cyclins . Because both CDK2 and p130Rbr-2 show affinity for cyclins, we suggest that p130Rbr-2 and CDK2 interacted through a yeast-derived cyclin bridge in the two-hybrid screen . The gene encoding p130Rbr-2 mapped to 16q13, a region of frequent genomic alteration in human tumors.

Mol Cell Biol, 1993 Dec, 13(12), 7232 - 8
Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis; Rapp WD et al.; Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides . Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W . Rapp and D . Stern, EMBO J . 11:1065-1073, 1992) . We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail . Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels . Some mutations, scattered throughout this region, have lesser effects or no effect . Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12 . Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

J Am Acad Dermatol, 1993 Dec, 29(6), 1008 - 12
A randomized, double-blind, placebo-controlled trial of ketoconazole 2% shampoo versus selenium sulfide 2.5% shampoo in the treatment of moderate to severe dandruff; Danby FW et al.; BACKGROUND: Ketoconazole is highly effective against the yeast Pityrosporum ovale, an organism believed to be involved in the pathogenesis of dandruff . OBJECTIVE: Our purpose was to evaluate the safety and effectiveness of ketoconazole 2% shampoo versus selenium sulfide 2.5% shampoo and placebo shampoo in patients with moderate to severe dandruff . METHODS: Features assessed included adherent and loose dandruff scores, presence or absence of irritation, itching, yeast cells, and global improvement rating by the investigator . RESULTS: A total of 246 patients were included . Mean total adherent dandruff score declined throughout the treatment period with both ketoconazole 2% and selenium sulfide 2.5% shampoos significantly better than placebo at all visits . Ketoconazole was statistically superior to selenium sulfide at day 8 only (p = 0.0026) . Both medicated shampoos were significantly better than placebo for reducing irritation and itching . Of the nine adverse experiences reported during the treatment phase, all involved patients treated with selenium sulfide 2.5% shampoo . CONCLUSION: Both ketoconazole 2% shampoo and selenium sulfide 2.5% shampoo are effective in the treatment of moderate to severe dandruff; however, ketoconazole 2% shampoo appears to be better tolerated.

Int J Dev Biol, 1993 Dec, 37(4), 589 - 94
Microinjection of suc1 transcripts delays the cell cycle clock in Patella vulgata embryos; Colas P et al.; The suc1 protein is a cell cycle regulator whose precise function remains to be elucidated . The suc1 cDNA of the mollusk Patella vulgata was cloned and sequenced . It encodes a 9 kD protein showing a strong similarity with its human counterparts and to a lesser extend with its yeast counterparts . The expression of suc1 in maturing oocytes was shown to be tightly cell cycle-regulated . The abundance of the suc1 transcripts is high in prophase- and metaphase-arrested oocytes but drops dramatically upon exit from M-phase, after fertilization . The microinjection of suc1 synthetic messengers into embryonic blastomeres delayed the cell cycle clock, thus disrupting the perfect cell cycle synchrony exhibited by the blastomeres of early Patella embryos . Interestingly, this suc1 delaying effect was significantly reversed when cyclin B messengers were co-injected with suc1 messengers . These results show that Patella embryos offer a quite valuable model to study cell cycle regulation . Moreover, they support the existence of a negative control exerted by suc1 on the cell cycle traverse in a higher eukaryote.

Int J Dev Biol, 1993 Dec, 37(4), 509 - 17
Identification of an amphibian oocyte nuclear protein as a candidate for a role in embryonic DNA replication; Bucci S et al.; Monoclonal antibody B24/3 recognizes a nuclear protein of 104 kD in germinal vesicles of newt oocytes . Immunohistostaining of oocytes at different stages of growth shows an accumulation of B24 protein throughout oogenesis . During development B24 protein is located inside embryonic cell nuclei from the onset of cleavage onwards . It gradually decreases from gastrulation and disappears at the tailbud stage . The NvB24 17.1 clone was isolated from an ovary expression library of the newt Notophthalmus viridescens and then sequenced: the open reading frame is capable of encoding a polypeptide of 744 amino acids . Northern blot experiments have shown that the 17.1 clone recognizes a single transcript of about 3 Kb in the ovary . In situ hybridization experiments showed that B24 mRNA transcription starts from previtellogenic oocytes, and is followed by the appearance and gradual accumulation of B24 protein in germinal vesicles of medium and large size oocytes . Keeping in mind the sequence similarity shown by the B24 protein to the mouse P1 protein as well as to the budding yeast Mcm3 and fission yeast cdc21 proteins, B24 protein can be speculated to play a role in the events of DNA replication during early amphibian embryogenesis . As B24 antigen is located in the sphere organelles both inserted on the lampbrush chromosomes and free in the oocyte nucleoplasm, an additional possible role of B24 protein could be related to assembling and/or storing snRNPs during oogenesis.

Biol Pharm Bull, 1993 Dec, 16(12), 1244 - 7
Photodynamic DNA strand breaking activities of acridine compounds; Iwamoto Y et al.; Induction of single strand breaks in DNA was assessed by the conversion of supercoiled closed circular plasmid DNA into the open circular form . Euflavine produced single-strand breaks following irradiation but not in the control maintained in the dark . The single strand breaking activity of photoactivated euflavine was found to be dose-dependent . The effective dose conversion 50% (ED50) of the closed circular DNA to the open circular form was 0.53 microM . A comparison of 8 acridine compounds revealed that the ED50 of diaminoacridines such as euflavine, proflavine and acridine yellow or the 3,6-dimethylamino-derivative (acridine orange) was less than 1 microM while the ED50 values of the other acridines were greater than 80 microM . Euflavine was markedly inhibited by singlet oxygen scavengers such as NaN3, histidine, alpha-tocopherol or beta-carotene and partly inhibited by superoxide dismutase, mannitol or catalase . These results suggest that enflavine induces single strand breaks in DNA mainly by a type II photodynamic mechanism . Photodynamic single strand breaking activities appeared related to their mutagenic activities on yeast . This experimental system described here is useful for the quantitative assessment of the single strand breaking activities of various photosensitizers in vitro and for the determination of active oxygen species involved in those processes.

Hum Mol Genet, 1993 Dec, 2(12), 2099 - 107
Isolation of a putative transcriptional regulator from the region of 22q11 deleted in DiGeorge syndrome, Shprintzen syndrome and familial congenital heart disease; Halford S et al.; A wide spectrum of birth defects are caused by deletions of the DiGeorge syndrome critical region (DGCR) at human chromosome 22q11 . Over one hundred such deletions have now been examined and a minimally deleted region of 300kb defined . Within these sequences we have identified a gene expressed during human and murine embryogenesis . The gene, named TUPLE1, and its murine homologue, encodes a protein containing repeated motifs similar to the WD40 domains found in the beta-transducin/enhancer of split (TLE) family . The TUPLE1 product has several features typical of transcriptional control proteins and in particular has homology with the yeast Tup1 transcriptional regulator . We propose that haploinsufficiency for TUPLE1 is at least partly responsible for DiGeorge syndrome and related abnormalities.

Hum Mol Genet, 1993 Dec, 2(12), 1995 - 9
Characterization of a methylation imprint in the Prader-Willi syndrome chromosome region; Dittrich B et al.; In adult human tissues, a HpaII and a CfoI restriction site at the PW71 (D15S63) locus in the Prader-Willi syndrome region on chromosome 15 are methylated on the maternal chromosome, but unmethylated on the paternal chromosome . The HpaII site is part of a sequence with high homology to the long terminal repeat of human endogenous retroviruses . Another HpaII site at the PW71 locus is methylated on both chromosomes . Sperm DNA carries the adult paternal methylation pattern . Oocyte DNA could not be studied . In chorion, placenta and tumor DNA, both HpaII sites are unmethylated . These findings suggest that the PW71 methylation imprint is established in the germline and that extraembryonic tissues and tumors are hypomethylated.

Hum Mol Genet, 1993 Dec, 2(12), 1991 - 4
Molecular definition of the Prader-Willi syndrome chromosome region and orientation of the SNRPN gene; Buiting K et al.; The Prader-Willi syndrome and the Angelman syndrome are caused by the loss of function of distinct but closely linked genes on human chromosome 15 . Based on a yeast artificial chromosome restriction map and two key patients we have determined that the shortest region of deletion overlap in the Prader-Willi syndrome comprises 320 kb . The region includes the anonymous DNA marker PW71 (D15S63) and the gene for the small nuclear ribonucleoprotein N (SNRPN) . The SNRPN gene maps 130 kb distal to PW71 and is transcribed from centromere to telomere.

Mamm Genome, 1993 Dec, 4(12), 687 - 94
Detailed physical and genetic mapping in the region of plasminogen, D17Rp17e, and quaking; Cox RD et al.; We present here a detailed physical map encompassing over 600 kb of mouse Chromosome (Chr) 17 in the region of plasminogen, D17Rp17e, and quaking . This region is cloned in yeast artificial chromosomes (YACs) . We have identified several CpG islands within this region from pulsed field gel mapping of mouse genomic DNA and YAC DNA . Five new DNA probes have been generated . One, D17Leh514, is a minimum of about 90 kb distal to plasminogen . Four, D17Leh513, D17Leh512, D17Leh511, and D17Leh510, are proximal to D17Rp17e, the closest previously described genetic marker to quaking viable and quaking lethal-1 mutations . We have genetically mapped D17Leh511 to within 0.15 cM of these mutations . The genetic distance to D17Rp17e from D17Leh511 is also 0.15 cM; the physical distance of less than 360 kb (minimum 200 kb) is consistent with an approximation of 2 Mbp per cM.

C R Acad Sci III, 1993 Dec, 316(12), 1484 - 8
{First generation of the physical map of the human genome}; Cohen D et al.; Sets of ordered overlapping cloned genomic fragments, spanning each of the human chromosomes are urgently needed for identification of human disease genes . Such a physical map provides also a unique source of material to study structure and function of the genome . To achieve this goal we exhaustively analyzed the CEPH yeast artificial chromosome (YAC) library containing 33,000 clones, which insert sizes were individually measured . With 0.9 Mb average lengths these YACs cover an equivalent of 10 haploid genomes . Then, several mapping techniques were combined to generate multiple structural information for most of these clones . Finally, the library was screened with more than 2,000 genetic markers quasi-uniformly distributed along 90% of the genome . These results should allow the scientific community to construct detailed chromosomic maps . Moreover, we propose a data analysis strategy that produces a first generation integrated map covering most of the human genome.

Nihon Kyobu Shikkan Gakkai Zasshi, 1993 Dec, 31 Suppl, 5 - 11
{Pathogenesis of summer-type hypersensitivity pneumonitis}; Ando M; Summer-type hypersensitivity pneumonitis (SHP) is the most prevalent type of HP in Japan, and the major causative agent of the disease is T . cutaneum . The major antigenic substance of the yeast is serotype-related polysaccharide . Home environmental factors indicate that SHP is a sick house syndrome . Immunologically, both humoral and cellular hypersensitivities are involved in the induction of the disease . The levels of specific IgG and IgA antibodies and complements in BAL fluids from SHP patients are well correlated to the clinical course . On the other hand, BAL lymphocytes in SHP patients are mostly T lymphocytes, mainly due to an increase in CD8+ subpopulations of lymphocytes; this leads to a decrease in the ratio of CD4+ to CD8+ . These T cells belong to CD45RO+ memory T lymphocytes and LFA-11 alpha high+ cytotoxic T lymphocytes as assessed by their cell surface phenotypes . However, functions of these BAL T lymphocytes remain undefined . Host factors such as HLA-DQw3 and cigarette smoking may participate in the development of the disease . In conclusion, the pathogenesis of SHP is considered to be a combination of immune complex disease and cellular hypersensitivity to T . cutaneum.

Hum Mol Genet, 1993 Dec, 2(12), 2051 - 4
The major peripheral myelin protein zero gene: structure and localization in the cluster of Fc gamma receptor genes on human chromosome 1q21.3-q23; Pham-Dinh D et al.; We have characterized the human gene encoding the major peripheral myelin protein zero (P0) and assigned it, by in situ hybridization, to the q21.3-q23 region of human chromosome 1 . This region is known to contain a cluster of interspersed genes coding for the related human leukocyte receptors of the Fc portion of the immunoglobulin G (Fc gamma RI, II, III) . This colocalization was refined by the finding of a yeast artificial chromosome (YAC) of the Centre d'Etude du Polymorphisme Humain (CEPH) library, hybridizing to the P0 and Fc gamma RIIA genes, demonstrating their physical linkage . These data may have important implications in demyelinating diseases studies like Charcot-Marie-Tooth disease type 1B (CMT1B).

Am J Physiol, 1993 Dec, 265(6 Pt 1), C1511 - 6
RNA subunit of mitochondrial RNA-processing enzyme is induced by contractile activity in striated muscle; Ordway GA et al.; A small RNA encoded within the nucleus of yeast and mammalian cells is an essential subunit of a mitochondrial RNA-processing endonuclease (RNase MRP) that generates primers for mitochondrial DNA (mtDNA) replication . We examined expression of MRP-RNA in specialized subtypes of mammalian striated muscles that differ markedly in respiratory activity and in muscles subjected to chronic stimulation via the motor nerve, a potent stimulus to mitochondrial biogenesis . MRP-RNA was more abundant in mitochondria-rich cardiac and slow-twitch skeletal muscles than in glycolytic fast-twitch skeletal muscles . Forced contractile activity resulting from nerve stimulation increased expression of MRP-RNA by 3.5-fold within the first day and by 14-fold within 14 days . Changes in abundance of MRP-RNA preceded but otherwise occurred in parallel to changes in specific activity of citrate synthase, a marker of mitochondrial proliferation shown previously to correlate with mtDNA copy number in this model . Another small RNA (U1) also was induced transiently (1-3 days) by nerve stimulation, but such changes were not sustained and were of less magnitude (< 4-fold) than changes in MRP-RNA . These findings are consistent with the hypothesis that MRP-RNA may have a regulatory function with respect to mtDNA replication and mitochondrial biogenesis.

Biochem Biophys Res Commun, 1993 Nov 30, 197(1), 154 - 62
Human elongation factor EF-1 beta: cloning and characterization of the EF1 beta 5a gene and assignment of EF-1 beta isoforms to chromosomes 2,5,15 and X; Pizzuti A et al.; We report here the isolation and characterization of a novel human elongation factor-1 beta (EF-1 beta) gene by cDNA selection from YAC mapping on chromosome 5q12-q14 . This gene is specifically transcribed in fetal brain and in skeletal muscle and is characterized by a complete sequence homology with previously described EF-1 beta cDNAs . We also assigned the loci for three other EF-1 beta isoforms, to human chromosomes 2, 15 and X . The multiple chromosomal assignments of EF-1 beta loci demonstrates the genetic heterogeneity of human EF-1 beta peptides.

FEBS Lett, 1993 Nov 29, 335(1), 73 - 5
RPII15 codes for the M(r) 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II; Liu Z et al.; The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II . Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies . Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence . Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster . This analysis identified the M(r) 15,000 subunit 9 as gene product of RPII15.

Cell, 1993 Nov 19, 75(4), 817 - 25
WAF1, a potential mediator of p53 tumor suppression; el-Deiry WS et al.; The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor . Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line . The WAF1 gene was localized to chromosome 6p21.2, and its sequence, structure, and activation by p53 was conserved in rodents . Introduction of WAF1 cDNA suppressed the growth of human brain, lung, and colon tumor cells in culture . Using a yeast enhancer trap, a p53-binding site was identified 2.4 kb upstream of WAF1 coding sequences . The WAF1 promoter, including this p53-binding site, conferred p53-dependent inducibility upon a heterologous reporter gene . These studies define a gene whose expression is directly induced by p53 and that could be an important mediator of p53-dependent tumor growth suppression.

Cell, 1993 Nov 19, 75(4), 753 - 63
Dif, a dorsal-related gene that mediates an immune response in Drosophila; Ip YT et al.; There are striking parallels between the regulation of gene expression along the dorsoventral (DV) axis of Drosophila embryos and lymphoid-restricted expression in the mammalian immune system . Both depend on regulatory factors containing rel domains (dorsal and NF-kappa B) that are controlled at the level of nuclear transport . A novel Rel-containing gene in Drosophila, Dif (dorsal-related immunity factor), provides a potential link between these seemingly disparate processes . Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae . Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury . Evidence is presented that once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes . These results suggest that mammalian and insect immunity share a common evolutionary origin.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1414 - 21
Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum; Romer S et al.; We have shown that both geranylgeranyl pyrophosphate synthase and phytoene synthase from pepper and tomato chromoplasts are very similar in terms of enzymic activity and immunological properties . This enabled us to clone cDNAs specific for phytoene synthase from ripening tomato and pepper fruits and to compare their amino acid sequences with those of bacterial phytoene synthase and yeast squalene synthase . Comparison of the nucleotide sequence of the 3'-untranslated region of the pepper phytoene synthase cDNA suggests that this region was subjected to complex recombination events . RNA gel blot hybridizations revealed that induction of phytoene synthase gene expression is much stronger in tomato than in pepper fruits . In contrast with tomato, 2 phytoene synthase transcripts of different size are present in pepper leaves and fruits . Comparison of the expression pattern of the genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase revealed that these genes are not co-regulated during pepper fruit ripening.

Eur J Biochem, 1993 Nov 15, 218(1), 261 - 5
Reconstitution of holotransketolase is by a thiamin-diphosphate-magnesium complex; Booth CK et al.; When human erythrocyte apo-transketolase is mixed with cofactors and substrates, the progress curve exhibits a lag phase . Elimination of the lag phase requires the presence of saturating concentrations of cofactors, thiamin diphosphate and Mg2+ . The most simple explanation of the observed hysteretic transition is that the slow binding of a Mg(2+)-thiamin-diphosphate species precedes slow isomerisation of the enzyme to the active form . Although the hysteretic transition involves more than one process, it does not involve the dissociation-association of enzyme subunits . The best estimate of the apparent Km, 1.59 +/- 0.23 microM, for the binding of Mg(2+)-thiamin diphosphate to transketolase was obtained in the presence of a high non-inhibitory concentration of magnesium and varied concentrations of thiamin diphosphate . Thus the reconstitution of the human enzyme differs from the yeast enzyme, which undergoes a rate-limiting dimerisation during reconstitution.

J Biol Chem, 1993 Nov 15, 268(32), 24099 - 105
A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana; Iba K et al.; Mutations at the fad7 locus of Arabidopsis thaliana (previously called fadD) cause decreased desaturation of dienoic fatty acids in chloroplast lipids in plants grown at elevated temperatures . This suggested that the fad7 locus encodes a chloroplast omega-3 desaturase that catalyzes the desaturation of lipid-linked 18:2 and 16:2 fatty acids . In order to clone the fad7 gene, it was first genetically mapped relative to the flanking restriction fragment length polymorphism markers 4547 and 2488A on chromosome 3, and yeast artificial chromosomes covering the locus were identified . A putative desaturase cDNA clone that was isolated by low stringency heterologous probing with a cDNA for an endoplasmic reticulum-localized omega-3 desaturase (fad3) hybridized to the yeast artificial chromosomes and could not be resolved from the locus by restriction fragment length polymorphism mapping . Expression of the cDNA in transgenic fad7 mutant plants resulted in restoration of wild type fatty acid composition and suppression of a previously observed effect of the fad7 mutation on chloroplast number, indicating genetic complementation . The structural gene contained seven introns within a coding sequence of 1338 base pairs, which encodes a 446-amino acid polypeptide of 51,172 daltons . The amino-terminal region of the fad7 gene product contained a consensus chloroplast transit peptide . Except for the amino-terminal domain, the deduced amino acid sequence of the fad7 gene product had high homology to the fad3 gene product, indicating that fad7 encodes an omega-3 desaturase and that the two genes arose from a common ancestral gene . There was no apparent effect of growth temperature on the steady-state levels of fad7 mRNA in wild type plants.

Gene, 1993 Nov 15, 133(2), 249 - 54
Cloning a pseudogene and cDNA encoding a 17-kDa ribosomal protein from mouse: structure and regulation of expression; Rudert F et al.; An rp lambda 5 cDNA encoding a ribosomal protein (r-protein) and a pseudogenic form of the corresponding gene (rp lambda 7) have been cloned from mouse . This cDNA codes for a highly basic protein of 160 amino acids (aa) with a deduced M(r) of 17,601, and most likely represents the species homolog of a recently cloned rat cDNA, which has been proposed to encode a homolog of the yeast r-protein, YL43 . The entire rp lambda 5 gene encompasses less than 1.5 kb of genomic DNA and apparently is composed of only two exons, as deduced from sequence comparison with its very similar pseudogenic variant, rp lambda 7 . Southern analysis, using the rp lambda 5 cDNA as a probe, indicates the existence of a great number of highly related sequences in the mouse genome . The mRNA for rp lambda 5 is approximately 800 nucleotides (nt) long and is found to be ubiquitously expressed at high levels in embryonic and adult mouse tissues, as shown by Northern and in situ analyses . Retinoic acid (RA) seems to have a moderate down-regulatory effect on this mRNA in differentiating P19 embryonal carcinoma cells . Several degenerate/nondegenerate RA-response element (RARE) motifs are found within 560 bp upstream from the degenerate start codon in rp lambda 7 . However, it is unknown whether this RA effect is exerted at the transcriptional and/or posttranscriptional levels.

FEBS Lett, 1993 Nov 15, 334(2), 149 - 52
A hybrid protein kinase-RNase in an interferon-induced pathway?
Bork P, Sander C.
The sequence of RNase L has been re-examined by computer analysis . We propose a molecular architecture of RNase L, with an unusual combination, in one protein chain, of 9 ankyrin-like repeats, a functional active protein kinase and a C-terminal catalytic RNase similar to the yeast protein, IRE1 . The protein kinase may be involved in a new signal transduction pathway which remains to be discovered.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10856 - 60
Myelin protein zero gene mutated in Charcot-Marie-tooth type 1B patients; Su Y et al.; Autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells . In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family . The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate . The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family {total multipoint logarithm of odds (lod) = 11.4 at theta = 0.00} with a splice junction mutation . Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago . MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes > 50% of myelin protein . These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5117 - 23
Coincidence painting: a rapid method for cloning region specific DNA sequences; Bailey DM et al.; We have developed a novel coincidence cloning strategy, termed Coincidence Painting, which enables the rapid generation of large numbers of region specific sequences . Coincidence Painting utilises Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification of flow sorted derivative translocation chromosomes . The PCR products are hybridised in situ onto specific flow sorted chromosomes for coincident sequence selection . Eluted and reamplified material is then cloned using a novel insert end revelation and ligation technique . Cloned inserts range in size from 150-1300 bps of which approximately 54% appear to be single copy sequences . The cloning method permits the excision of vector free probe for library hybridisation screening and the small insert size facilitates analysis for the generation of sequence tagged sites (STSs) . We have used such clones successfully for YAC screening by PCR and for cosmid screening by filter hybridisation . This new methodology should allow the rapid saturation with probes of regions defined by specific translocation breakpoints.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5034 - 40
Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease; Puchta H et al.; Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast . We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies . We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences . We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay . GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections . The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells . At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction . These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.

Lancet, 1993 Nov 6, 342(8880), 1131 - 4
Treatment of active Crohn's disease by exclusion diet: East Anglian multicentre controlled trial; Riordan AM et al.; Elemental diet is as effective in producing remission of Crohn's disease (CD) as is corticosteroid treatment, but most patients relapse soon after resumption of a normal diet . We have investigated the efficacies of dietary modification and oral corticosteroids in maintaining remission achieved with elemental diet . In a multicentre trial, 136 patients with active CD were started on elemental diet and other treatment was withdrawn . 43 (31%) declined to continue elemental diet for 14 days, but 78 (84%) of the remaining 93 achieved remission and were randomly assigned corticosteroids (38) or diet (40) . Corticosteroid treatment started at 40 mg prednisolone daily, which was tapered and stopped after 12 weeks; that group received dietary advice on healthy eating . The diet group received "tapered" placebo and were instructed to introduce one new food daily, excluding any that precipitated symptoms . Assessment of progress for up to 2 years was made by physicians unaware of group assignment . Intention-to-treat analysis showed median lengths of remission of 3.8 (interquartile range 5.0) months in the corticosteroid group and 7.5 (15.3) months on diet, and relapse rates at 2 years, adjusted for withdrawals, of 79% and 62%, respectively (p = 0.048) . Clinical improvement in the diet group was associated with significant changes in plasma albumin and alpha 1-antichymotrypsin concentrations and erythrocyte sedimentation rate . Food intolerances discovered were predominantly to cereals, dairy products, and yeast . Diet provides a further therapeutic strategy in active Crohn's disease.

J Biol Chem, 1993 Nov 5, 268(31), 23311 - 7
Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase; LaVallie ER et al.; Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys . In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase . The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis . We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus . Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2 . The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide . The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.

Plant Cell, 1993 Nov, 5(11), 1591 - 8
Potato sucrose transporter expression in minor veins indicates a role in phloem loading; Riesmeier JW et al.; The major transport form of assimilates in most plants is sucrose . Translocation from the mesophyll into the phloem for long-distance transport is assumed to be carrier mediated in many species . A sucrose transporter cDNA was isolated from potato by complementation of a yeast strain that is unable to grow on sucrose because of the absence of an endogenous sucrose uptake system and the lack of a secreted invertase . The deduced amino acid sequence of the potato sucrose transporter gene StSUT1 is highly hydrophobic and is 68% identical to the spinach sucrose transporter SoSUT1 (pS21) . In yeast, the sensitivity of sucrose transport to protonophores and to an increase in pH is consistent with an active proton cotransport mechanism . Substrate specificity and inhibition by protein modifiers are similar to results obtained for sucrose transport into protoplasts and plasma membrane vesicles and for the spinach transporter, with the exception of a reduction in maltose affinity . RNA gel blot analysis shows that the StSUT1 gene is highly expressed in mature leaves, whereas stem and sink tissues, such as developing leaves, show only low expression . RNA in situ hybridization studies show that the transporter gene is expressed specifically in the phloem . Both the properties and the expression pattern are consistent with a function of the sucrose transporter protein in phloem loading.

Protein Eng, 1993 Nov, 6(8), 939 - 46
Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties; Bakir U et al.; Nine single amino acid mutations in the active site of Aspergillus awamori glucoamylase were made by cassette mutagenesis to alter the pH dependence of the enzyme and to determine possible functions of the mutated residues . The Glu179-->Asp mutation expressed in yeast led to a very large decrease in kcat but to no change in Km, verifying this residue's catalytic function . Asp176-->Glu and Glu180-->Asp mutations affected Km more than kcat, implying that Asp176 and Glu180 are involved in substrate binding or structural integrity . The Leu177-->Asp mutation decreased kcat only moderately, probably by changing the position of the general acid catalytic group, and did not affect Km . The Trp178-->Asp mutation greatly decreased kcat while increasing Km, showing the importance of Trp178 in the active site . Val181-->Asp and Asn182-->Asp mutations changed kinetic values little, suggesting that Val181 and Asn182 are of minor catalytic and structural importance . Finally, insertions of Asp or Gly between residues 176 and 177 resulted in almost complete loss of activity, probably caused by destruction of the active site structure . No large changes in pH dependence occurred in those mutations where kinetic values could be determined, in spite of the increase in most cases of the total negative charge . Increases in activation energy of maltoheptaose hydrolysis in most of the mutant glucoamylases suggested cleavage of individual hydrogen bonds in enzyme-substrate complexes.

J Cell Sci, 1993 Nov, 106 ( Pt 3), 789 - 802
The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A; Roa M et al.; Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells . Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution . By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells . BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours . The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center . The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence . Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble . Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures . Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride . Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

J Am Coll Health, 1993 Nov, 42(3), 111 - 5
Sexually transmitted diseases in college men: a preliminary clinical investigation; Sawyer RG et al.; Continuing efforts to educate college students about the dangers of unprotected sexual intercourse have resulted in little evidence of positive change in sexual behavior . This clinical study examined the sexual behavior, perceived risk of human immunodeficiency virus, and pathology of 66 university men attending a health center's men's clinic for treatment of sexually transmitted disease (STD) . The study demonstrated the existence of a high-risk group of men who, despite sexually transmitted disease pathology, high numbers of sexual partners, inconsistent condom use, and delays in seeking treatment, perceive their risk of contracting HIV/AIDS as being extremely low . This preliminary investigation suggests the need for specific education interventions and future in-depth studies of this populationPIP: A reduction in the prevalence of sexually transmitted diseases (STD) suggests that sexually active individuals are taking more precautions to prevent infection during sexual intercourse . Accordingly, male college students presenting for their first time at a men's STD clinic at a large East Coast public university were examined and surveyed . Their individual pathologies, behavioral characteristics, and perceived risk of HIV infection were identified . 13 graduate students, 23 seniors, 25 juniors, 4 sophomores, and 1 freshman were seen during the fourth weeks of Spring and Fall semesters in 1992 . 26 men (39.4%) reported having 7 or more lifetime sex partners, yet only 9 (13.6%) reported always using condoms; 19 (28.8%) reported never using condoms . 33 were currently in relationships lasting more than 1 year, 16 were in recent relationships of up to 6 months, 13 were in casual relationships, 2 had multiple partners, and 2 reported having no sex partners . 31 reported previously visiting other STD clinics, among who 23 had been diagnosed with an STD . Only 20 of the young men visited the clinic within 7 days of developing symptoms, even though symptoms included genital lesions (39.4%) and rashes (28.8%) . 23 (34%) were diagnosed with human papilloma virus, 8 with nongonococcal urethritis, 7 with general dermatological problems, 5 with testicular problems, 4 with yeast infection, and 4 with molloscum contagiosum . 9 men visited the clinic to have questions answered and 6 had no discernible pathology . None of the subjects perceived themselves as being at extremely high risk of contracting HIV, 4 thought themselves at high risk, 12 were neutral about their risk, 37 felt at low risk, and 13 felt at extremely low risk . Educational interventions and in-depth studies of the population are needed .

Genomics, 1993 Nov, 18(2), 223 - 9
ZNF75: isolation of a cDNA clone of the KRAB zinc finger gene subfamily mapped in YACs 1 Mb telomeric of HPRT; Villa A et al.; We have previously mapped a zinc finger genomic motif (ZNF75) to the Xq26 cytogenetic band by using a hybrid panel . Here, we report the isolation of the transcribed counterpart in a cDNA clone and its further localization . The cDNA clone, from a lung fibroblast library, is assembled from three exons, including a 289 amino acid (AA) long open reading frame containing a recently described motif, the Kruppel-associated box, 42 AA long, in exon 2 . By comparison with other reported members of the subfamily, the exon-intron boundaries also appear to be very well conserved . Further analysis allowed us to map this gene 1 Mb downstream from the HPRT gene in the published YAC contig that extends across Xq26 . Two other motifs, 87 and 78% homologous to ZNF75 at the amino acid level, were identified by PCR on total human DNA, but map outside Xq24-qter.

Pediatr Res, 1993 Nov, 34(5), 565 - 71
Immunomodulating actions of nucleotides: enhancement of immunoglobulin production by human cord blood lymphocytes; Jyonouchi H et al.; We have shown previously that polynucleotides enhance in vitro antibody and Ig production in response to T-dependent antigens in mice and augment Ig production by adult human peripheral blood mononuclear cells . Herein, we report their effects on umbilical cord blood mononuclear cells (CBMNC) obtained from full-term babies . CBMNC produced much less IgM/IgG and an almost negligible amount of IgA in response to various stimuli compared with adult peripheral blood mononuclear cells . The supplementation of yeast RNA augmented spontaneous and T-dependent IgM (p < 0.01) but not IgG production by CBMNC . This action was largely attributable to polynucleotides, which appeared to exert their actions in a dose-dependent manner at the initial stages of culture . Their actions were dependent upon the presence of T cells, but they also enhanced spontaneous IgM production by CBMNC in the absence of T cells . Preincubation of T cells from CBMNC and peripheral blood mononuclear cells with RNA for 3 h before the culture resulted in enhanced IgM production, independent of the stimulants used . Thus, polynucleotides appear to exert actions on immature human T cells as well as other lineage cells in vitro . Their actions may be dependent on the presence or absence of antigens or other stimuli and the nature of the stimuli (T dependent versus T independent) . These findings may further support the potential importance of nucleotides contained in human breast milk.

Hum Mol Genet, 1993 Nov, 2(11), 1901 - 5
Regional assignment of 19 X-linked ESTs; Parrish JE et al.; Subchromosomal localizations for 19 X-linked expressed sequence tags (ESTs) have been determined . Two ESTs are located in Xq28, adding two novel genes to this disease-rich region . The remaining ESTs are located primarily in the pericentromeric region, with most mapping to Xp11.1-p21.1 . YAC and cosmid genomic clones have been isolated for several of these loci . Available cDNAs have been used to characterize the corresponding transcripts by Northern analysis in multiple human tissues.

Hum Mol Genet, 1993 Nov, 2(11), 1765 - 72
The utrophin and dystrophin genes share similarities in genomic structure; Pearce M et al.; Utrophin and dystrophin are highly homologous proteins which are reciprocally expressed in DMD (Duchenne muscular dystrophy) muscle . The remarkable similarity of these proteins suggests that they may play a similar cellular role in some circumstances; if this were the case then utrophin may be capable of replacing dystrophin in DMD patients . In this paper we show that the genomic structure of the utrophin gene is similar to the dystrophin gene, further exemplifying the relatedness of the two genes and their gene products . We have constructed a 1.25 Mb contig of eight yeast artificial chromosome (YAC) clones covering the utrophin gene located on chromosome 6q24 . Utrophin is encoded by multiple small exons spanning approximately 900 kb . The distribution of exons within the genomic DNA has similarities to that of the dystrophin gene . In contrast to dystrophin, the utrophin gene has a long 5' untranslated region composed of two exons and a cluster of unmethylated, rare-cutting restriction enzyme sites at the 5' end of the gene . Similarities between the genomic structure suggest that utrophin and dystrophin arose through an ancient duplication event involving a large region of genomic DNA.

Mamm Genome, 1993 Nov, 4(11), 662 - 9
A physical map of the human APP gene in YACs; Rooke K et al.; Several point mutations within exons 16 and 17 of the amyloid precursor protein (APP) gene have been reported that are associated with Alzheimer's disease in a small number of familial cases . To determine the size of the APP gene and the organization of the exons within human genomic DNA, we have characterized 11 Yeast Artificial Chromosome (YAC) recombinants containing human APP gene sequences . The smallest YAC insert was 125 kb, and the largest was 1.4 Mb . The YACs were screened by polymerase chain reaction amplification of APP exons to determine which of the 18 exons coding for APP770 were present . Four of the YACs (D110G1, D110G6, D110E9, and B142F9) contain all 18 exons and at least part of the promoter . Construction of an overlapping map of the gene with all of the YACs demonstrated that 3 of the 11 YACs were chimeric . The orientation and position of the coding sequence on the map was determined by probing digests of the YAC DNA with exon PCR products and the vector arms . The coding region of the APP gene spans approximately 400 kb of genomic DNA.

Mol Biol Evol, 1993 Nov, 10(6), 1215 - 26
Molecular phylogeny and evolutionary rates of alcohol dehydrogenases in vertebrates and plants; Yokoyama S et al.; Phylogenetic relationships and evolutionary rates for 36 alcohol dehydrogenases (ADHs) from vertebrates and plants are described, with ADHs from fission yeast and from baker's yeasts as outgroups . Vertebrate sequences include 15 mammalian, 2 avian, and 1 amphibian ADH, as well as one sequence deduced from a human pseudogene . Plant ADH sequences include 1 from a gymnosperm (loblolly pine) and 16 from angiosperms, in which 9 sequences are from monocots and 7 are from dicots . Phylogenetic analysis shows that ADHs from vertebrates and from plants are classified into two distinct groups, and the latter group is further divided into angiosperm and gymnosperm ADHs . Among three classes of vertebrate ADHs, class I and II ADHs are most closely related and evolved at much faster rates than did class III ADHs . The gymnosperm ADH has evolved more slowly than any angiosperm ADH . However, ADHs within both of the vertebrate classes I and II and class III groups have similar evolutionary rates, as do most of the ADHs within the angiosperm group . The rate of amino acid replacement for the vertebrate and plant ADHs is approximately 0.3-0.6 x 10(-9)/site/year.

Cancer Genet Cytogenet, 1993 Nov, 71(1), 15 - 21
Cloning and characterization of the human t(3;6)(p14;p11) translocation breakpoint associated with hematologic malignancies; Smith SE et al.; The t(3;6)(p14;p11) chromosome translocation was identified in a family in which three members developed hematologic malignancies . To help characterize the region on chromosome 3 surrounding this translocation breakpoint, two flanking lambda clones, MS156 and MJ1525, were linked by pulsed-field gel electrophoresis to the same 510-kb NotI fragment on chromosome 3 . MS156 was localized to a region proximal to the breakpoint of a der(3) chromosome somatic cell hybrid (derived from the t(3;6) cell line), and MJ1525 localized distal to the breakpoint . MJ1525 was used to screen the CEPH yeast artificial chromosome (YAC) library, which revealed a YAC, 195F3, that spanned the breakpoint . Subcloning into Lambda DASH II and production of a contiguous array of overlapping lambda clones revealed a clone, L17, that spanned the breakpoint . A rare restriction endonuclease map for the YAC 195F3 was constructed, and multiple clusters of rare restriction sites within the YAC were identified, possibly indicating the disruption of a gene by the t(3;6) translocation breakpoint.

Am J Clin Nutr, 1993 Nov, 58(5), 649 - 52
Selenium status of lactating women is affected by the form of selenium consumed; McGuire MK et al.; The impact of providing selenomethionine (2.7 mumol Se) or selenium-enriched yeast (2.9 mumol Se) on the selenium status of lactating and nonlactating women with customary intakes of approximately 1.3 mumol Se/d was studied . Plasma selenium declined in unsupplemented lactating women but not in nonlactating women . Selenomethionine increased plasma selenium in both lactating and nonlactating women whereas selenium-enriched yeast increased plasma selenium only in nonlactating women . Erythrocyte selenium concentration was not significantly modified by lactation . Plasma glutathione peroxidase (GPx) activity decreased with duration of lactation in unsupplemented women and selenomethionine or selenium-enriched yeast supplementation prevented the decline . Milk selenium declined markedly for 20 wk after parturition in unsupplemented women . Selenomethionine significantly increased milk selenium concentrations whereas selenium-enriched yeast prevented a decline . These results clearly show that the source of selenium provided to lactating women can significantly influence selected indexes of selenium status, including milk selenium concentration.

Am J Clin Nutr, 1993 Nov, 58(5), 643 - 8
Selenium status of infants is influenced by supplementation of formula or maternal diets; McGuire MK et al.; Plasma selenium of infants fed proprietary formula was significantly less than that in infants fed human milk . Addition of selenite to the formula (0.253 mumol Se/L) increased plasma selenium and activities of glutathione peroxidase (GPx) and total peroxidase (Px) . However, erythrocyte selenium decreased significantly during the 12-wk study in infants receiving human milk or formula with or without supplemental selenite . Infants fed human milk from women receiving 0 or 200 micrograms supplemental selenium as selenomethionine or selenium-enriched yeast had plasma selenium that paralleled changes in their selenium intake . Plasma GPx and Px activities were unrelated to human milk selenium intake . Milk from women given either selenium supplement prevented the decline in infant erythrocyte selenium . Results of these studies suggest that the method of feeding modifies the infant's apparent selenium status and that the molecular form of selenium provided and/or its interaction with other milk constituents are determinants of infant selenium status.

Toxicol Appl Pharmacol, 1993 Nov, 123(1), 34 - 42
Cytochrome P4501A1 mediates the metabolism of 2,3,7,8-tetrachlorodibenzofuran in the rat and human; Tai HL et al.; Previous studies have established that TCDF is rapidly metabolized and excreted in rats and that pretreatment of rats with TCDD increases the rate of hepatic metabolism of this compound . The extrahepatic metabolism of TCDF was investigated to assess which enzyme was involved in the metabolism of this compound . Very little metabolism of TCDF was detected in control microsomes (0.3-3.0 pmol/mg/hr), while TCDF metabolism was increased 40- to 200-fold in TCDD-induced rat liver, kidney, and lung microsomes . Since TCDD induces cytochrome P4501A1 and P4501A2 (CYP1A1 and CYP1A2) in the rat liver but only CYP1A1 in kidney and lung, these results suggest that CYP1A1 metabolizes TCDF . To test this hypothesis, TCDF metabolism was investigated in the presence and absence of selective chemical inhibitors and antibodies to CYP1A1 and 1A2 . 1-Ethynylpyrene, a suicide inhibitor of CYP1A1 and antibody to rat CYP1A1, produced a dose-dependent inhibition of TCDF metabolism in TCDD-induced rat liver microsomes . Conversely, 2-ethynylnaphthalene, a suicide inhibitor of CYP1A2 and antibody to rat CYP1A2, had no inhibitory effect on the hepatic microsomal metabolism of TCDF . Together, the results strongly indicate that rat CYP1A1 is the primary enzyme responsible for the metabolism of TCDF . 4-Hydroxy-2,3,7,8-TCDF was also identified as the major TCDF metabolite formed by rat CYP1A1 . TCDF was also metabolized by human liver microsomes and recombinant yeast microsomes expressing human CYP1A1 and reductase but not by yeast microsomes expressing human CYP1A2 with or without reductase . A similar HPLC profile of TCDF metabolites was observed with microsomes from human liver and yeast expressing human CYP1A1 . However, based on ethoxyresorufin-O-deethylase activity, a marker of CYP1A1, the relative rate of TCDF metabolism is about 100-fold greater in TCDD-induced rat liver microsomes than in yeast microsomes expressing human CYP1A1 and reductase . Thus, although TCDF is metabolized by rat and human CYP1A1, the results indicate that there are marked quantitative differences in metabolism which suggest that TCDF will be more persistent in humans.

Scand J Immunol, 1993 Nov, 38(5), 423 - 7
Impaired phagolysosomal fusion of peripheral blood monocytes from HIV-infected subjects; Pittis MG et al.; We evaluated phagolysosomal fusion in peripheral blood monocytes from 20 HIV-infected individuals and 40 normal controls, using a fluorescence assay with acridine orange as marker . The percentages of phagolysosomal fusion of monocytes from HIV-infected subjects, after 30 and 60 min of yeast ingestion, (mean +/- standard deviation) 57.2 +/- 17 and 63.2 +/- 18.6, respectively, when compared to normal controls (72.4 +/- 7.8 and 77 +/- 8.1), did not differ significantly . However, there was a direct linear association between the percentages of phagolysosomal fusion and CD4+ lymphocytes (P < 0.001) or CD4/CD8 T-cell ratio (P < 0.01) . These results suggest that phagolysosomal dysfunction becomes evident at late stages of HIV infection and progresses as CD4+.T-lymphocyte count and CD4/CD8 T-cell ratio decrease . On the other hand, recombinant gp120 inhibited significantly normal phagolysosomal fusion at concentrations ranging between 1 and 1000 ng/ml . Taking together the results obtained, we can conclude that gp120 could be responsible for monocyte phagolysosomal dysfunction observed in HIV infected patients.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10375 - 9
Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system; Takacs AM et al.; Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system . Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT) . The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region . Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other . Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain . Phosphorylation of P protein was not required for N-P association . Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P . This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.

Genes Dev, 1993 Nov, 7(11), 2235 - 45
Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex; Yokomori K et al.; The basal factor TFIIA has been shown to act early during initiation in both the mammalian and yeast transcription systems, but a TFIIA-like activity has not been identified in Drosophila . While characterizing the Drosophila TFIID complex, we discovered that a 30-kD protein that cofractionated with dTFIID was homologous to the previously identified, large subunit of yeast TFIIA . Here, we report the cloning and biochemical characterization of Drosophila TFIIA-L . Coimmunoprecipitation studies with anti-dTBP, anti-dTFIIA-L, and anti-TAF antibodies indicated a tight association of the endogenous dTFIIA and dTFIID . However, dTFIIA could be dissociated from dTFIID under conditions that did not elute the TAFs, and the eluted material had mobility shift and transcriptional activities associated with TFIIA . Peptide sequence and Western analysis with antibodies raised against the amino- and carboxy-terminal portions of recombinant dTFIIA-L revealed that a precursor 48-kD species was cleaved in vivo, giving rise to the 30- and 20-kD subunits of dTFIIA that remain associated with each other and with dTFIID . Protein-protein interaction assays identified dTBP and dTAFII110 as targets for binding TFIIA in the TFIID complex . These results suggest that TFIIA may form a specific complex with both TAFs and other components of the transcriptional machinery during formation of the initiation complex.

Eur J Immunol, 1993 Nov, 23(11), 2860 - 7
The human immunoglobulin kappa locus . Characterization of the partially duplicated L regions; Huber C et al.; The L regions are parts of the C kappa proximal (p) and distal (d) copies of the human immunoglobulin kappa locus and are therefore called the Lp and Ld regions . The two regions with their 25 V kappa genes and pseudogenes have now been cloned, thus completing the cloning of the kappa locus . Lp has been linked to the neighboring Ap and B regions, while Ld was linked to Ad . There is good evidence that at the other side of Ld, i.e . towards the centromere, the end of the locus has been reached . Most of the cloning and linking was achieved by chromosomal walking, employing cosmid and phage lambda clones . No such clones could be found for three small gaps . Two of them were closed by a polymerase chain reaction strategy; the third one was characterized by genomic blot hybridization experiments and eventually bridged by a yeast artificial chromosome clone . Early in evolution, a stretch of about 25 kb which comprised three V kappa genes near the 5' end of the L region precursor must have been duplicated, such that the later duplication of large parts of the kappa locus resulted in the appearance of two very similar three-gene regions in each, Lp and Ld . Two deletions in the central parts of the L regions, on the other hand, must have occurred after the duplication of the locus, since they are found in Lp and Ld in different positions.

Development, 1993 Nov, 119(3), 673 - 90
A Drosophila G1-specific cyclin E homolog exhibits different modes of expression during embryogenesis; Richardson HE et al.; We have isolated a Drosophila homolog of the human G1-specific cyclin E gene . Cyclin E proteins thus constitute an evolutionarily conserved subfamily of metazoan cyclins . The Drosophila cyclin E gene, DmcycE, encodes two proteins with a common C-terminal region and unique N-terminal regions . Unlike other Drosophila cyclins, DmcycE exhibits a dynamic pattern of expression during development . DmcycE is supplied maternally, but at the completion of the cleavage divisions and prior to mitosis 14, the maternal transcripts are rapidly degraded in all cells except the pole (germ) cells . Two modes of DmcycE expression are observed in the subsequent divisions . During cycles 14, 15 and 16 in non-neural cells, DmcycE mRNA levels show no cell-cycle-associated variation . DmcycE expression in these cells is therefore independent of the cell cycle phase . In contrast, expression in proliferating embryonic peripheral nervous system cells occurs during interphase as a brief pulse that initiates before and overlaps with S phase, demonstrating the presence of a G1 phase in these embryonic neural cell cycles . DmcycE appears not to be expressed in cells that undergo endoreplication cycles during polytenization . The structural homology to human cyclin E, the ability of DmcycE to rescue a G1 cyclin-deficient yeast strain, the presence of multiple PEST sequences characteristic of G1-specific cyclins and expression during G1 phase in proliferating peripheral nervous system cells all argue that Drosophila cyclin E is a G1 cyclin . Constitutive DmcycE expression in embryonic cycles lacking a G1 phase, in contrast to expression during the G1-S phase transition in cycles exhibiting a G1 phase, implicates DmcycE expression in the regulation of the G1 to S phase transition during Drosophila embryogenesis.

Breast Cancer Res Treat, 1993 Nov, 28(2), 121 - 35
Molecular cloning of BRCA1: a gene for early onset familial breast and ovarian cancer; Bowcock AM; Molecular analyses allow one to determine genetic lesions occurring early in the development of tumors . With positional cloning approaches we are searching for a gene involved in the development of early onset familial breast and ovarian cancer that maps to human chromosome 17q21 and is termed BRCA1 . This involves localizing the region genetically within families with multiply affected members, capturing the region identified by genetic analyses in YACs (yeast artificial chromosomes), converting those YACs to smaller manipulable pieces (such as cosmids), and searching for genes via a variety of approaches such as direct screening of cDNA libraries with genomic clones, direct selection by hybridization, "exon trapping", and CpG island rescue . Once identified, candidate genes will be screened for mutations in affected family members in whom breast cancer segregates with the locus on 17q21 . The frequency of this gene has been calculated to be 0.0033; from this the incidence of carriers, i.e . those carrying such a predisposition, is one in 150 women . The isolation of BRCA1 and the elucidation of the mutations resulting in breast and ovarian cancer predisposition will allow identification of women who have inherited germ-line mutations in BRCA1 . In families known to harbor a germ-line BRCA1 mutation, diagnosis of affected members will be rapid . It is possible that one will also be able to detect alterations of the second copy of this gene early in tumor development in individuals carrying a germ-line mutation . It is not yet known how frequently somatic BRCA1 mutations predispose to breast and ovarian carcinoma in the general female population . If, as in other genetic diseases, new germ-line mutations occur in some women and thus contribute to the development of breast cancer, it may be feasible to screen women in the general population for predisposing mutations . In addition, if acquired genetic mutations of the BRCA1 gene are involved as early events in the development of non-familial forms of the disease, early detection of possible breast carcinoma may become feasible in biopsy of breast tissue.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1993 Nov, 13(11), 667 - 9, 645
{Mechanism of guizhi tang on dual-directional thermoregulation--effect of prostaglandin E2 level in hypothalamus of rats}; Fu HY et al.; Antipyretic action was found in febrile rats induced by yeast, and body temperature was elevated in hypothermia rats induced by aminopyrine, when 10 g/kg of Guizhi Tang (GZT) was administered per os . The content of prostaglandin E in the hypothalamus of rats was determined with the radioimmunoassay . Administration of GZT in 10 g/kg p.o . could both decrease the PGE2 level of hypothalamic blood in febrile rats, and increase the PGE2 level in hypothermia rats . Antipyretic action of GZT was also displayed when microinjection of PGE2 into the lateral ventricle which produced fever in rats . The evidence was provided that GZT might carry out at least part of the dual-directional regulating action in body temperature through the promotion or inhibition on PGE2 synthesis, release, or metabolism in the thermoregulatory center.

Mol Biochem Parasitol, 1993 Nov, 62(1), 83 - 92
Characterization of the pfmdr2 gene for Plasmodium falciparum; Zalis MG et al.; We report the characterization of the pfmdr2 gene which is a gene related to the P-glycoprotein family but with a somewhat different structure than the mdr genes . Based on DNA sequence analysis of genomic clones, we have discovered that the pfmdr2 gene has 10 predicted transmembrane domains and a single ATP-binding site . In a homology search using GenBank sequences, we discovered that the pfmdr2 gene has a significant homology with the hmt1 gene in yeast . The yeast hmt1 gene is involved in cadmium resistance and is hypothesized to transport cadmium containing complexes from the cell . We have further characterized the pfmdr2 gene expression by northern analysis and discovered that it is expressed in a stage-specific manner, only at the trophozoite stage and not in ring stages . We have prepared a rabbit antibody to a recombinant fusion protein expressing a portion of the pfmdr2 coding region . In IFA analysis, this antibody stains trophozoites and not ring stages . Western analysis reveals a protein of approximately 110 kDa which is consistent with the size of the predicted open reading frame based on DNA sequence analysis . Based on this analysis and previous work, there is no evidence for a change in pfmdr2 expression in drug-resistant versus drug-sensitive parasites.

Mycoses, 1993 Nov-Dec, 36(11-12), 445 - 8
Phaeohyphomycosis caused by Exophiala jeanselmei treated with itraconazole; Schwinn A et al.; Phaeohyphomycotic cysts developed on the right knee of a 72-year-old woman undergoing immunosuppressive treatment for ulcerative colitis 6 years after accidental inoculation of soil in a bicycle accident . The lesions were red, firm, slightly raised, 0.5-1 cm in size and completely asymptomatic . The diagnosis was made by histopathological examination of three excised cysts and by repeated isolation of Exophiala jeanselmei in pure culture . The excised cyst walls contained large numbers of dematiaceous fungal elements in the form of hyphae, yeast-like cells and some cells dividing internally by a transverse septum . The patient was treated with 200 mg of itraconazole daily, but the treatment had to be stopped because of severe side-effects after 6 weeks . Histologically the cysts were cleared of dematiaceous elements, but E . jeanselmei could still be isolated from one of two skin biopsies 1 month after the end of therapy.

J Biochem (Tokyo), 1993 Nov, 114(5), 714 - 7
The complete amino acid sequence of subunit d of rat liver mitochondrial H(+)-ATP synthase; Higuti T et al.; Subunit d of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high performance liquid chromatography . The partial amino acid sequence of the subunit was determined by automated Edman degradation of the peptide fragments . The nucleotide sequence of subunit d of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA . The sequence was composed of 581 nucleotides including a coding region for the import precursor of subunit d and noncoding regions on the 5'- and 3'- sides . The possible precursor of subunit d and its mature polypeptide deduced from the open reading frame consisted of 161 and 160 amino acid residues with molecular weights of 18,763 and 18,631, respectively . Subunit d is a hydrophilic protein with an isoelectric point of 6.19 . The sequence of the rat subunit d is highly homologous with that of subunit d of bovine heart and slightly similar to that of the subunit d of the yeast mitochondria . However, it had no homology with the sequence of any of the subunits of bacterial or chloroplast H(+)-ATP synthase.

Mol Biol Evol, 1993 Nov, 10(6), 1370 - 9
Phylogenetic relationships of reverse transcriptase and RNase H sequences and aspects of genome structure in the gypsy group of retrotransposons; Springer MS et al.; The gypsy group of long-terminal-repeat retrotransposons contains elements having the same order of enzyme domains in the pol gene as do retroviruses . Elements in the gypsy group are now known from yeast, filamentous fungi, plants, insects, and echinoids . Reverse transcriptase and RNase H amino acid sequences from elements in the gypsy group--including the recently described SURL elements, TED, Cft1, and Ulysses,--were aligned and analyzed by using parsimony and bootstrapping methods, with plant caulimoviruses and/or retroviruses as outgroups . Clades supported at the 95% level after bootstrapping include (1) 17.6 with 297 and (2) all of the SURL elements together . Other likely relationships supported at lower bootstrap confidence intervals include (1) SURL elements with mag, (2) 17.6 and 297 with TED, and this collective group with 412 and gypsy, (3) Tf1 with Cft1, (4) IFG7 with Del, and (5) all of the retrotransposons in the gypsy group together, to the exclusion of Ty3 . In contrast with an earlier analysis, our results place mag within the gypsy group rather than outside of a cluster that contains gypsy group retrotransposons and plant caulimoviruses . Several features of retrotransposon genomes provide further support for some of the aforementioned relationships . The union of SURL elements with mag is supported by the presence of two RNA binding sites in the nucleocapsid protein . Location of the tRNA primer binding site and the presence of a long open reading frame 3' to the pol gene support the 17.6-297-TED-412-gypsy cluster.

J Biol Chem, 1993 Oct 25, 268(30), 22920 - 6
DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active; Bojanowski K et al.; Immunoprecipitation of DNA topoisomerase II from yeast results in a preparation that contains casein kinase II; this suggests that the two proteins may associate in the intact cell . Purified recombinant topoisomerase II and casein kinase II associate to form a complex in vitro which is stable after topoisomerase II becomes phosphorylated by the kinase . Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit alone can efficiently phosphorylate topoisomerase II, the formation of a stable topoisomerase II-casein kinase II association requires the presence of the beta subunit of the kinase . Both proteins engaged in this complex retain their catalytic activities . Naturally occurring polyamines and polyanionic compounds appear to be crucial factors governing the interaction between the two proteins . Although the biological significance of a stable catalytically active topoisomerase II-casein kinase II molecular complex remains to be defined, these observations suggest the possibility of a novel mechanism regulating topoisomerase II and casein kinase II activities.

J Comp Neurol, 1993 Oct 22, 336(4), 571 - 82
Presence and biological activity of a GnRH-like factor in the nervous system of Helisoma trivolvis; Goldberg JI et al.; Gonadotropin-releasing hormone (GnRH) constitutes a family of neuropeptides found throughout the vertebrates . Although a GnRH-like peptide has also been isolated from yeast (alpha-mating factor), the presence of GnRH has not been clearly demonstrated in invertebrate phyla . In this study, we tested the hypothesis that GnRH-like peptides are present and functional in the central nervous system (CNS) of the gastropod mollusc, Helisoma trivolvis . The presence of a GnRH-like peptide was examined by three methods: (1) in immunofluorescence studies with four different antibodies generated against several GnRH peptides, select neurons and putative neurosecretory cells were specifically and consistently labelled throughout the CNS; (2) reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) analysis revealed a GnRH-like factor which co-migrates with mammalian (m)GnRH; and (3) in bioactivity experiments, extracts of Helisoma trivolvis CNS mimicked GnRH in stimulating gonadotropin release from dispersed goldfish pituitary cells in static culture . Two functional assays were carried out to examine the potential biological roles of GnRH-like peptides in Helisoma . (1) Intracellular recordings of left-parietal and visceral ganglion neurons revealed diverse electrophysiological responses to mGnRH . These effects were attenuated by a mGnRH antagonist . (2) Addition of mGnRH arrested neurite outgrowth in a subpopulation of dissociated embryonic Helisoma neurons in culture . Taken together, these results strongly suggest that a mGnRH-like peptide is an important neuropeptide in Helisoma . A hypothesis is presented that GnRH-like peptides may be ancient factors that are conserved both structurally and functionally in the evolution of animals.

Biochim Biophys Acta, 1993 Oct 19, 1216(1), 65 - 72
Molecular cloning of cDNAs for two Xenopus proteasome subunits and their expression in adult tissues; Fujii G et al.; Proteasome, a large protein complex with ATP-dependent protease activities, is composed of non-identical but closely related multi-subunits . Using cDNAs for rat proteasome subunits as probes, we obtained three cDNA clones for two Xenopus proteasome subunits from ovary cDNA library . The primary structures of the three cDNAs showed high homology to the corresponding proteasome subunits of other mammalian species (above 90%) and also considerable homology to those of Drosophila and yeast . These results indicate that the sequences of proteasome subunits are well conserved during evolution . Northern blot hybridization revealed that RNAs for the newly isolated subunits (XC8 and XC9) and the previously isolated subunit (XC3) occur at very high levels in testis and ovary, at moderately high levels in lung, skin kidney and spleen, and at low levels in liver, stomach and muscle . It was also shown that relative amounts of the mRNAs for the three subunits are similar in all the adult tissues examined . From these results, we concluded that the expression of the genes for the three subunits (XC3 XC8 and XC9-1) takes place in a roughly coordinated manner in different adult tissues.

Biochim Biophys Acta, 1993 Oct 19, 1216(1), 129 - 33
A highly conserved repetitive sequence from Physarum polycephalum contains nucleotide arrangements similar to replicator sequences; Kruse L et al.; An interspersed repetitive sequence from Physarum polycephalum has been cloned and analysed . The 394 bp sequence is highly conserved and contains several homopolymeric (dA)-(dT) tracts capable of forming bent DNA structures and a 10/11 match to the yeast-ARS-consensus sequence . The repetition frequency of the described sequence is about 3000 to 7000, a number that would fit with the distribution of replicator segments in Physarum.

Biochim Biophys Acta, 1993 Oct 19, 1216(1), 119 - 22
Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin; Turley SJ et al.; We have isolated and determined the sequence of a 1.1-kb cDNA from a murine WEHI-3 macrophage library which encodes the highly conserved, nucleolar protein, fibrillarin . The murine fibrillarin protein sequence displays 94.2% identity with human fibrillarin, 82.9% identity with amphibian fibrillarin and 74.0% identity with the yeast fibrillarin homolog, NOP1 . Immunoprecipitation showed that anti-fibrillarin autoantibodies from human scleroderma sera and the monoclonal autoantibody 72B9 recognize the approx . 34-36 kDa in vitro transcribed and translated protein . Mouse fibrillarin contains a N-terminal glycine- and arginine-rich (GAR) domain which although conserved among the fibrillarins is not as strongly conserved as several regions in the carboxy tail of the protein . Specific amino acid residues in yeast NOP1 thought to be associated with the synthesis and maturation of ribosomes show strong conservation between the mouse, human, amphibian and yeast protein sequences.

Biochim Biophys Acta, 1993 Oct 19, 1216(1), 113 - 4
The primary structure of two proteins from the small ribosomal subunit of rice; Nishi R et al.; We isolated two rice cDNAs which encode an open reading frame of 117 amino acids or 82 amino acids . Their deduced amino acid sequence correspond to the ribosomal proteins (r-protein) . A comparison of the amino acid sequence shows that the deduced amino acid sequence of one cDNA is homologous to the rat r-protein S20 and Xenopus r-protein S22 . Another encoded products with a high degree of homology to the rat r-protein S21 and the yeast r-protein YS25.

Biochim Biophys Acta, 1993 Oct 19, 1216(1), 110 - 2
The primary structure of two proteins from the large ribosomal subunit of rice; Nishi R et al.; We isolated two rice cDNAs which encode an open reading frame of 389 amino acids . Their deduced amino acid sequence corresponded to the ribosomal protein (r-protein) . A comparison of amino acid sequence shows that the deduced amino acid sequence of one cDNA is homologous to Arabidopsis, yeast and the rat r-protein L3 . Another encoded products with a high degree of homology to the rat r-protein L7A and yeast r-protein L4.

Biochemistry, 1993 Oct 19, 32(41), 11204 - 10
Characterization of isomers of monoamminechromium-ATP and their use in mapping enzyme active sites; Rawlings J et al.; Twelve isomers formed by the reaction of monoamminechromium(III) with ATP have been synthesized . Isomerism in this system results from chirality around the beta-phosphorus of the ATP, the position of the ammonia ligand, the relative orientation of the ammonia and the AMP, and the presence of ring-puckering conformers . By using chromatography on cross-linked cycloheptaamylose, reverse-phase C-18 HPLC, and cation-exchange FPLC, these isomers have been separated and purified . Their structures have been identified by (1) cleavage by periodate, followed by elimination in the presence of diethylenetriamine and subsequent phosphate insertion to give lambda, delta, or meso facial monoamminechromium tripolyphosphate with molar ellipticities of +240, -240, or 0 deg cm2 dmol-1 at 550 nm, respectively, (2) cleavage by nucleotide pyrophosphatase to give meridional or facial monoamminechromium pyrophosphate, (3) spectral data, and (4) rates of interconversion of isomers . All possible isomers are seen except those with ammonia syn to AMP . Since the substitution of ammonia for water in the inner coordination sphere appears to diminish affinity for enzymes when the ammonia is in contact with the protein but not when it faces the solvent, these isomers are useful for mapping of enzyme active sites . Their use as probes of enzyme structure is illustrated by their behavior with yeast hexokinase.

Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9408 - 12
Reproducing the three-dimensional structure of a tRNA molecule from structural constraints; Major F et al.; The three-dimensional structure of yeast tRNA(Phe) was reproduced at atomic resolution with the automated RNA modeling program MC-SYM, which is based on a constraint-satisfaction algorithm . Structural constraints used in the modeling were derived from the secondary structure, four tertiary base pairs, and other information available prior to the determination of the x-ray crystal structure of the tRNA . The program generated 26 solutions (models), all of which had the familiar "L" form of tRNA and root-mean-square deviations from the crystal structure in the range of 3.1-3.8 A . The interaction between uridine-8 and adenosine-14 was crucial in the modeling procedure, since only this among the tertiary pairs is necessary and sufficient to reproduce the L form of tRNA . Other tertiary interactions were critical in reducing the number of solutions proposed by the program.

Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9247 - 51
Raf-1 protein kinase activates the NF-kappa B transcription factor by dissociating the cytoplasmic NF-kappa B-I kappa B complex; Li S et al.; Addition of mitogenic growth factors to quiescent cells triggers complex signal transduction cascades that result in the reprogramming of gene expression and entry into the cell cycle . We have found that an oncogenic variant of the c-Raf-1 protein kinase stimulated the expression of promoters containing NF-kappa B binding sites . In situ immunofluorescence analysis revealed elevated nuclear levels of the p65 subunit of NF-kappa B in v-raf-transformed NIH 3T3 cells . Incubation of HeLa cell cytoplasmic extracts with a purified recombinant glutathione S-transferase-raf fusion protein in the presence of ATP released active NF-kappa B that could be detected by electrophoretic gel mobility shift assay . Coincubation of purified recombinant I kappa B and glutathione S-transferase-raf in the presence of ATP resulted in the phosphorylation of I kappa B . Coexpression of GAL4 (activation domain)-I kappa B and GAL4 (DNA-binding domain)-raf fusion proteins in yeast resulted in stimulation of a GAL4-responsive reporter gene, indicating that I kappa B and Raf interact physically in vivo . These results indicate that the Raf-1 kinase functions in signal transduction in part by activating the NF-kappa B transcription factor by phosphorylating I kappa B in the cytoplasmic I kappa B-NF-kappa B complex to release active NF-kappa B.

Cancer Genet Cytogenet, 1993 Oct 15, 70(2), 99 - 102
Characterization of two marker chromosomes in a patient with acute nonlymphocytic leukemia by two-color fluorescence in situ hybridization; Taniwaki M et al.; A patient with acute nonlymphocytic leukemia (ANLL), M5b according to French-American-British (FAB) classification, showed monosomy 16, an extra 1p-, and a 21q+ . These derivative chromosomes could not be defined by GTG-banding . For better characterization, we performed two-color fluorescence in situ hybridization (FISH) experiments applying DNA libraries from sorted human chromosomes, chromosome-specific repetitive probes, and a band-specific YAC-clone . With these FISH studies the karyotype could be characterized as 46,XY, +der(1)t(1;21)(p11;?), -16,der(21)t(16;21) (p11.1;q22).

Biochem J, 1993 Oct 15, 295 ( Pt 2), 607 - 9
Does phosphoglucoisomerase display anomeric specificity or selectivity towards alpha-D-glucose 6-phosphate? An assessment by two-dimensional phase-sensitive 31P exchange spectroscopy (EXSY) n.m.r; Kayser F et al.; The study by two-dimensional phase-sensitive 31P exchange spectroscopy (EXSY) n.m.r . of hexose 6-phosphates interconversion in the reaction catalysed by yeast phosphoglucoisomerase reveals that the enzyme displays anomeric selectivity, rather than specificity, towards alpha-D-glucose 6-phosphate . Indeed, beta-D-glucose 6-phosphate participates for about 20% to the total and direct conversion of the aldohexose into oxohexose ester.

Eur J Biochem, 1993 Oct 15, 217(2), 535 - 40
Molecular cloning and primary structure of Man9-mannosidase from human kidney; Bause E et al.; Man9-mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide-bound Man9-GlcNAc2 oligosaccharides producing a single Man6 isomer {Bause, E., Breuer, W., Schweden, J., Roesser, R . & Geyer, R . (1992) Eur . J . Biochem . 208, 451-457} . We have isolated four Man9-mannosidase-specific clones from a human kidney cDNA library and used these to construct a full-length cDNA of 3250 base pairs . A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies . Analysis of the coding sequence predicts that Man9-mannosidase is a type II transmembrane protein consisting of a short cytoplasmic polypeptide tail, a single transmembrane domain acting as a non-cleavable signal sequence and a large luminal catalytic domain . This domain architecture closely resembles that of other ER and Golgi-located processing enzymes, pointing to common structural motifs involved in membrane insertion and topology . The protein sequence of the Man9-mannosidase contains three potential N-glycosylation sites of which only one site is used . The amino acid sequence of several peptide regions, including a calcium-binding consensus sequence, bears striking similarities to an ER alpha-1,2-mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER alpha-mannosidase and Golgi alpha-mannosidase II . This finding may indicate that the mammalian alpha-mannosidases, which differ significantly in their substrate specificity, are coded for by evolutionarily unrelated genes, providing an attractive means of regulation and fine-tuning oligosaccharide processing, not only at the enzymic but also at the transcriptional level.

Nucleic Acids Res, 1993 Oct 11, 21(20), 4783 - 7
A method for the generation of YAC transgenic mice by pronuclear microinjection; Schedl A et al.; Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size . The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation . Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes . Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA . As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated . Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations . YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice . Applications and benefits of this technique will be discussed.

Nucleic Acids Res, 1993 Oct 11, 21(20), 4769 - 76
Isolation and characterization of the binding sequences for the product of the Arabidopsis floral homeotic gene AGAMOUS; Huang H et al.; The Arabidopsis floral homeotic gene AGAMOUS (AG) is required for normal flower development . The deduced AG protein contains a region which shares substantial sequence similarity with the DNA-binding domains of known transcription factors, SRF (human) and MCM1 (yeast) . Therefore, it is likely that AG is also a DNA-binding protein regulating transcription of floral genes . We describe here several experiments to characterize AG-DNA binding in vitro . We show that AG indeed binds a DNA sequence matching the consensus of SRF targets . Further, we have selected the AG-binding sequences from a pool of random oligonucleotides, and deduced an AG-binding consensus sequence of TT(A/T)CC(A/T)(A/t)2(T/A)NNGG(-G)(A/t)2 . We have demonstrated that AG binds to the consensus region of three of the oligonucleotides by footprinting analysis . Finally, we have examined AG's relative binding affinity for different sequences, as compared to SRF, by gel mobility shift analysis . Our results indicate that AG is a sequence-specific DNA-binding protein, and that the AG-binding consensus sequence is similar to those of MCM1 and SRF.

Biochim Biophys Acta, 1993 Oct 7, 1179(1), 11 - 22
Intracellular translocation of a 28 kDa GTP-binding protein during osmotic shock-induced cell volume regulation in Dunaliella salina; Memon AR et al.; The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina . We confirmed the previous identification by Rodriguez et al . (Rodriguez Rosales, M.P., Herrin, D.L . and Thompson, G.A., Jr . (1992) Plant Physiol . 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx . 21, 28, 28.5, 29 and 30 kDa . These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella . The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein . Immunodetection analysis showed that the D . salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein . This antibody was utilized for quantitative GTP-binding protein measurements as described below . Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D . salina 28-kDa protein but also the 29-kDa component . The 30-kDa GTP-binding protein of D . salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody . None of the D . salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins . When D . salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative {32P}GTP binding and {125I}antibody binding on nitrocellulose blots . This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold . No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Oct 5, 268(28), 21044 - 52
A novel CDC2-related protein kinase from Leishmania mexicana, LmmCRK1, is post-translationally regulated during the life cycle; Mottram JC et al.; The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle . We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box . lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level . lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant . These data suggest that Lmmcrk1 is unlikely to be the functional L . mexicana cdc2 homologue . A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms . SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box . These data suggest that a family of CDC2-related protein kinases are present in Leishmania . Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 8952 - 6
Selective amplification of additional members of the ADP-ribosylation factor (ARF) family: cloning of additional human and Drosophila ARF-like genes; Clark J et al.; The ADP-ribosylation factor (ARF) family is one of four subfamilies of the RAS superfamily of low molecular weight GTP-binding proteins (G proteins) . Highly degenerate oligonucleotides encoding two conserved regions were used in a PCR reaction to amplify cDNAs encoding each of the known ARF proteins and eight additional cDNA fragments encoding previously unreported human members of the ARF family . Additional sequences were obtained from yeast or fly libraries by using this technique . These oligonucleotides specifically amplify members of the ARF family but not the structurally related G protein alpha subunits or members of the other three subfamilies of the RAS superfamily . Fragments obtained by PCR were used to obtain full-length sequences encoding highly homologous ARF-like (ARL) gene products from human and Drosophila melanogaster libraries, termed ARL2 and Ar184F, respectively . The encoded proteins are each 184 amino acids long and are 76% identical, with 40-45% identity to human ARF1 and Drosophila arf-like (arl) proteins . These genes appear to be generally expressed in human tissues and during Drosophila development . The purified human ARL2 protein differed in several biochemical properties from human ARF proteins, including the complete absence of ARF activity . Thus, the ARF family of low molecular weight GTP-binding proteins includes at least 15 distinct but structurally conserved members, including both the functionally conserved ARF proteins and the functionally disparate ARL proteins . The latter proteins currently comprise two distinct gene products in Drosophila (arl and ARL84F) and one in man (ARL2).

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 8832 - 6
Interaction of human thyroid hormone receptor beta with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone; Baniahmad A et al.; The human thyroid hormone receptor beta (hTR beta) is capable of both transcriptional silencing and hormone-dependent activation . However, the detailed mechanism of this transcriptional regulation remains to be elucidated . One possibility is that hTR beta interacts directly with factors of the basal transcriptional machinery, thereby modulating basal promoter activity in a direct manner, as has been shown for other transcription factors . Here, we show that hTR beta interacts specifically with the human basal transcription factor TFIIB . Deletion analysis revealed two contact sites in the receptor: one is located in the N terminus, while the other is part of the ligand-binding domain (LBD) and is located at the C terminus . Interestingly, each receptor contact site interacts with different sites in TFIIB . Cotransfection experiments revealed that, when fused to the DNA-binding domain of yeast transcription factor GAL4, the C-terminal interaction site of hTR beta was transcriptionally inactive; however, when it was cotransfected with the remaining part of the LBD on a separate molecule, silencing function was restored . In agreement with that, we show that thyroid hormone is able to significantly decrease the interaction of its receptor LBD with TFIIB . Our data suggest that hTR beta acts as a transcriptional silencer by interacting with TFIIB and that thyroid hormone may act in part by preventing transcriptional repression at this level.

J Cell Biol, 1993 Oct, 123(1), 35 - 45
Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane; Huber LA et al.; Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells . We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products . Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane . Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface . Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic . Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected . We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.

Neuron, 1993 Oct, 11(4), 657 - 72
Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system; Buckanovich RJ et al.; Paraneoplastic opsoclonus-ataxia, a disorder of motor control, develops in breast or lung cancer patients who harbor an antibody (Ri) that recognizes their tumors and a nuclear neuronal protein of 55 kd . We have characterized a gene, Nova, encoding an antigen recognized by the Ri antibody . Nova encodes a novel, highly conserved protein, homologous to the RNA-binding protein hnRNP K, the yeast splicing protein MER1, and a motif in several retroviral proteases . Northern blot analysis detects Nova transcripts only in brain, and several alternatively spliced forms are present in brain and tumor cells . Nova expression is restricted to the ventral brain stem and spinal cord in E18 mice . Since Nova encodes a target antigen in the motor disorder paraneoplastic opsoclonus-ataxia that is expressed in the developing subcortical motor system, it is a likely participant in both the pathogenesis of paraneoplastic opsoclonus-ataxia and the developmental biology of the motor system . The homology between Nova and hnRNP K suggests that Nova regulates RNA splicing or metabolism in a specific subset of developing neurons.

Comput Appl Biosci, 1993 Oct, 9(5), 511 - 5
PFGE MAPPER: a tool to aid in the analysis of pulse field gel electrophoresis maps; Shifman MA; Pulse field gel electrophoresis mapping is an important technique for characterizing large segments of DNA and constructing long-range restriction maps . We have developed a tool, PFGE MAPPER, to aid in the construction of pulse field electrophoresis gel maps . This tool helps construct pulse field gel maps from single and double digest experiments visualized by hybridization with single copy probes . The program is written in Think C and runs on Macintosh computers . An intuitive interface allows the user to interactively modify fragment sizes or errors, select fragments for analysis and recalculate the maps . Maps can be printed or saved for later viewing . After constructing and saving several maps in a region, PFGE MAPPER can be used to refine and extend the overall map by merging individual maps . This tool should be useful for constructing long-range restriction maps of genomic DNA and yeast artificial chromosomes.

Med Hypotheses, 1993 Oct, 41(4), 308 - 15
Insulin resistance in Mexican Americans--a precursor to obesity and diabetes?
McCarty MF.
Mexican Americans appear to have a strong genetic predisposition to insulin resistance, android obesity, and type II diabetes, apparently as a function of Native American genetic heritage . Theoretical considerations suggest that insulin resistance may be a primary factor that plays a causative role in the induction of both obesity and diabetes . Measures which promote optimal insulin sensitivity--chromium picolinate, brewer's yeast, soluble fiber supplements, metformin, very-low-fat diet, exercise training--may have value for preventing, treating, or retarding the onset of obesity and diabetes, and merit clinical evaluation in this regard . Correction of insulin resistance may also lessen cardiovascular risk, in part by reducing LDL cholesterol and improving risk factors associated with Syndrome X . These comments are likely to be valid for other Native American groups at high risk for diabetes.

J Med Virol, 1993 Oct, 41(2), 95 - 8
Accelerated schedule of hepatitis B vaccination in patients with hemophilia; Santagostino E et al.; Early development of immunity after hepatitis B vaccination is particularly important for patients such as hemophiliacs, at high risk for acquiring hepatitis B from potentially infectious plasma-derived concentrates . The purpose of this study was to evaluate whether or not protective antibody titers could be achieved quickly and maintained in hemophiliacs by an accelerated vaccination schedule . A yeast-recombinant hepatitis B vaccine (Engerix B, SKF Ritt) was given subcutaneously in the deltoid region and repeated 2 and 6 weeks later to 85 hemophiliacs negative for hepatitis B virus (HBV) markers . After the first 22 patients had been enrolled, a modification of the schedule involving a fourth booster dose 24 weeks after the first dose of vaccine was applied to the next 63 consecutive vaccines . Fifty-three percent of vaccinees had antibody titers to hepatitis B surface antigen (anti-HBs > or = 10 mlU/ml) by week 6, even though the mean titers of anti-HBs were somewhat lower than those achieved historically in normal individuals . The protection rate had increased to 87% by week 10, one month after the third dose of vaccine, and to 93% by week 24 . One year after starting vaccination, the rate for the vaccinees who did not receive the fourth booster dose was 71%, and 96% for those who did receive the fourth dose, with only 2 patients not responding despite the booster dose . It is concluded that even though the accelerated schedule of immunization produced rapidly high rates of protective antibody titers, a booster dose is required to obtain higher titers and provide more persistent immunity.

Br J Haematol, 1993 Oct, 85(2), 406 - 8
A trial of recombinant human superoxide dismutase in patients with Fanconi anaemia; Liu JM et al.; Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia . The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage . Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA . Human SOD (rh-SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast . We treated four FA patients with a 2-week infusion of rh-SOD (25 mg/kg d daily) to determine whether rh-SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype . We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh-SOD treatment in two patients and that bone marrow progenitors were increased in one patient.

Eur J Cell Biol, 1993 Oct, 62(1), 49 - 58
Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium; Bengtsson T et al.; The role of changes in cytosolic free calcium concentration ({Ca2+}i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated . Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions . Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT) . In parallel, local {Ca2+}i changes were monitored using a fura-2 ratio imaging system . In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in {Ca2+}i, both around the phagosome and in the cell body . During continued phagocytosis, {Ca2+}i was more elevated around the phagosome compared to the rest of the cell . No {Ca2+}i fluctuations were observed in MAPT cells . Adhesion and phagocytosis led to a several-fold increase in F-actin . The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils . A distinct ring of F-actin was formed around a phagosome with a yeast particle . Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells . The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion . This treatment reestablished the periphagosomal {Ca2+}i rises, as observed in CCM cells . In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in {Ca2+}i, whereas the subsequent depolymerization is . The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of {Ca2+}i changes (Jaconi et al., J . Cell Biol . 110, 1555-1564 (1990)) . This suggests that the actin network, controlled by {Ca2+}i, regulates the movement of granules during phagocytosis.

Hum Mol Genet, 1993 Oct, 2(10), 1679 - 85
Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA); Thomas NS et al.; A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region . The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line . This map subdivides the region into at least 6 mapping-intervals . DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones . Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint . These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones . Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs . Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region . One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA . This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified . A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Mol Genet, 1993 Oct, 2(10), 1667 - 72
Physical mapping and YAC-cloning connects four genetically distinct 4qter loci (D4S163, D4S139, D4F35S1 and D4F104S1) in the FSHD gene-region; Wijmenga C et al.; We have constructed a long-range restriction map of the region on chromosome 4q that contains the gene for facioscapulohumeral muscular dystrophy (FSHD) . This region contains the linkage group cen .. . D4S163-D4S139-D4F35S1-D4F104S1-FSHD .. . 4qter, which spans a genetic distance of about 5 cM . Pulse field gel electrophoresis (PFGE) mapping indicated that these loci span a region not more than 1 Mb . STSs were developed for several of these loci, which served to isolate four overlapping yeast artificial chromosomes (YACs) . These YACs confirmed the PFGE map and have allowed us to generate a more detailed restriction map using cosmid contig mapping . The physical distances were smaller than was expected on the basis of the genetic map . Two potential HTF islands have been detected within the cloned region . One HTF island maps about 100 kb centromeric from the tandem repeats involved in the FSHD mutation, whereas the other maps within these tandem repeats.

Hum Mol Genet, 1993 Oct, 2(10), 1527 - 34
Cloning the breakpoint cluster region of the inv(16) in acute nonlymphocytic leukemia M4 Eo; Dauwerse JG et al.; The pericentric inversion of chromosome 16 and the t(16;16) are two recurrent aberrations in bone marrow of patients with acute nonlymphocytic leukemia subtype M4 Eo, characterized by abnormal eosinophilic granulation . We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread over the short arm of chromosome 16 and the detection, isolation and characterization of a 14Kb EcoRI fragment containing a cluster of breakpoints . First, cosmids were mapped to intervals defined by constitutional 16p rearrangements, second, the inv(16) and t(16;16) breakpoints were mapped to one of the intervals using FISH with the mapped cosmids and third, cosmids within this interval were ordered using two color interphase FISH . An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) breakpoints and one t(16;16) breakpoint analysed . In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint . Since this patient has the same phenotype, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo . This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to the 16q breakpoint . While four of the five YACs showed instability of the region around the inv(16) breakpoint, DNA halo analysis allowed us to identify one YAC which was co-linear with normal genomic DNA and has yielded the actual breakpoint sequences which could be subcloned into cosmids and fosmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Mikrobiyol Bul, 1993 Oct, 27(4), 352 - 6
{Incidence of oral candidiasis in diabetic patients}; Ozturkcan S et al.; Yeast infections play an important role in diabetics . Therefore poorly controlled diabetics and diabetics who have high levels of serum glucose carry a high risk . In the present study, 52 diabetics aged between 17-75 years old have been investigated by means of the incidence of oral candida and have been compared with control group of 33 healthy individuals . In poorly controlled diabetics, the rate of candida growth found high . It is also observed that this ratio is higher in type I than type II, and candida growth rate increased as well as serum glucose level . All of these results are found to be statistically important.

Nat Genet, 1993 Oct, 5(2), 195 - 200
Localization of Friedreich ataxia phenotype with selective vitamin E deficiency to chromosome 8q by homozygosity mapping; Ben Hamida C et al.; Friedreich ataxia and ataxia with selective vitamin E deficiency (AVED) share very similar clinical phenotypes . We have mapped the AVED locus to proximal 8q with only three large consanguinous Tunisian families, representing to our knowledge the first use of homozygosity mapping for primary linkage analysis . Subsequently, three additional families showed linkage with the same markers . A maximum lod score of 17.9 was obtained at theta = 0 for the haplotype D8S260-D8S510, consisting of the two closest markers . With only 6 families, the AVED locus is therefore mapped precisely as illustrated by the lod-1 confidence interval of 2.4 cM on either side of D8S260-D8S510 . Isolation of a yeast artificial chromosome contig > 800 kilobases (kb) showed that D8S260 and D8S510 are less than 400 kb apart.

Anal Biochem, 1993 Oct, 214(1), 128 - 34
The use of tributylphosphine and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in the study of protein sulfhydryls and disulfides; Chin CC et al.; The use of the reagent tributyl phosphine (Bu3P) to reduce disulfides (Ruegg, U.T., and Rudinger, J., Methods Enzymol . 47, 111-116, 1977) and of 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to block free sulphydryl groups (Toyo'oka, T., and Imai, K . Anal . Chem . 56, 2461-2464, 1984) is well established in the literature . Since the two reagents apparently do not react with each other, their combination offers a convenient and quite general method for the complete characterization of free Cys (SH) and crosslinked Cys (S-S) in proteins (Kirley, T.L., J . Biol . Chem . 264, 7185-7192, 1989) . We review some of the characteristics of the reaction of these reagents with Cys in peptides and proteins and some of the properties of the ABD-Cys derivatives . The review includes reactions with model compounds (e.g., Cys and glutathione), proteins such as enolase from yeast and rabbit muscle, containing only free Cys, a protein, fetuin, containing only crosslinked Cys, and a protein, superoxide dismutase, containing both free and crosslinked Cys . In all cases the direct comparison of the tryptic or chymotryptic peptides derived from the products of parallel reactions of the protein with ABD-F alone and with ABD-F together with Bu3P permitted the determination of both free and total Cys in the protein . Sequencing the fluorescent ABD-peptides established the position of the Cys residues in the primary sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Biochem, 1993 Oct, 214(1), 116 - 23
An examination of the reliability of the radiochemical assay for monoamine oxidases A and B; Krueger MJ et al.; The radiochemical assay for MAO A has been compared with the polarographic and alcohol dehydrogenase-coupled assays, using 5-hydroxytryptamine (5-HT) and tyramine and the homogeneous human liver enzyme expressed in yeast, and rat liver mitochondria . Despite efforts to measure true initial rates and to avoid known sources of error in the radiochemical procedure, significantly higher rates (Vmax) and lower Km values for the substrate were obtained with the polarographic than with the radiochemical method for 5-HT and tyramine, using either highly purified enzyme or mitochondria . The rate of tyramine oxidation measured by the polarographic method and the coupled assay agreed well, however . Consequently, for kinetic and inhibition studies we recommend the polarographic method for MAO A substrates . Comparison of the radiochemical, polarographic, spectrophotometric, and coupled assays for MAO B from beef liver (mitochondria and homogeneous enzyme) showed polarography to be the method of choice on all substrates tested, although, if all known sources of error are carefully controlled, the radiochemical assay gives the same Vmax and Km values . We also report that with each substrate studied the apparent Km is much lower when mitochondria are used than when the highly purified, virtually lipid-free preparations of MAO A are studied.

Lab Invest, 1993 Oct, 69(4), 396 - 404
Pulmonary granuloma formation in the rat is partially dependent on monocyte chemoattractant protein 1; Flory CM et al.; BACKGROUND: We have examined the role of MCP-1 (monocyte chemoattractant protein 1; also known as monocyte chemotactic and activating factor or the murine JE gene product) in the pathogenesis of glucan-induced granulomatous vasculitis in the rat . While in vitro studies indicate that MCP-1 possesses monocyte chemotactic and activating activities, little is known about its biologic role in pathologic processes . Glucan-induced pulmonary granulomatous vasculitis is an ideal model in which to study the role of MCP-1, because the granulomas develop rapidly and synchronously and are monocyte/macrophage-rich . EXPERIMENTAL DESIGN: The purpose of this study was to define the topographic distribution and temporal pattern of MCP-1 expression in the lungs of rats with evolving glucan-induced granulomatous vasculitis and to determine the effect of neutralization of MCP-1 activity on granuloma formation . Glucan-induced pulmonary granulomatous vasculitis was induced in rats by the intravenous infusion of yeast cell wall glucan . At the indicated time points after glucan infusion, rats were sacrificed and the lungs processed for Northern, immunohistochemical and in situ hybridization analyses of MCP-1 production . Morphometric analysis was used to quantify the effect of neutralization of MCP-1 activity on granuloma formation . RESULTS: Granuloma formation was accompanied by a biphasic increase in steady-state whole lung MCP-1 mRNA levels that peaked at 1 and 6 to 24 hours . In situ hybridization and immunohistochemical analyses revealed that components of the bronchial and vascular walls are responsible for the early rise (1 hour) in MCP-1 mRNA and protein expression, whereas granuloma-associated alveolar macrophages are the predominant source of MCP-1 later (6 to 24 hours) in the evolution of these lesions . Intravenous infusion and/or intratracheal instillation of neutralizing concentrations of anti-rat MCP-1 antibody raised against recombinant rat MCP-1 resulted in a dramatic decrease in the number and size of glucan-induced granulomas as well as in the numbers of mononuclear phagocytes retrieved in bronchoalveolar lavage fluid . CONCLUSIONS: These studies demonstrate that glucan-induced granulomatous vasculitis is accompanied by increased local expression of MCP-1 mRNA and protein, that there is a coordinated production of MCP-1 by different cell types within the lung during evolving glucan-induced pulmonary vasculitis, and that MCP-1 plays a requisite role in pulmonary granuloma formation.

J Med Genet, 1993 Oct, 30(10), 818 - 21
Isolation of a new marker and conserved sequences close to the DiGeorge syndrome marker HP500 (D22S134); Wadey R et al.; End fragment cloning from a YAC at the D22S134 locus allowed the isolation of a new probe HD7k . This marker detects hemizygosity in two patients previously shown to be dizygous for D22S134 . This positions the distal deletion breakpoint in these patients to the sequences within the YAC, and confirms that HD7k is proximal to D22S134 . In a search for coding sequences within the region commonly deleted in DGS we have identified a conserved sequence at D22S134 . Although no cDNAs have yet been isolated, genomic sequencing shows a short open reading frame with weak similarity to collagen proteins.

J Pharmacol Exp Ther, 1993 Oct, 267(1), 331 - 40
Complementary and synergistic antinociceptive interaction between the enantiomers of tramadol; Raffa RB et al.; The explanation for the co-existence of opioid and nonopioid components of tramadol-induced antinociception appears to be related to the different, but complementary and interactive, pharmacologies of its enantiomers . The (+) enantiomer had Ki values of only 1.33, 62.4 and 54.0 microM at mu, delta and kappa receptors, respectively . The (-) enantiomer had even lower affinity at the mu and delta sites (Ki = 24.8, 213 and 53.5 microM, respectively . The (+) enantiomer was the most potent inhibitor of serotonin uptake (Ki = 0.53 microM) and the (-) enantiomer was the most potent inhibitor of norepinephrine uptake (Ki = 0.43 microM) . Basal serotonin release was preferentially enhanced by the (+) enantiomer and stimulation-evoked norepinephrine release was preferentially enhanced by the (-) enantiomer . The (+) and (-) enantiomers each independently produced centrally mediated antinociception in the acetylcholine-induced abdominal constriction test (ED50 = 14.1 and 35.0 micrograms i.t., respectively) . Racemic tramadol was significantly more potent (P < .05) than the theoretical additive effect of the enantiomers (antinociceptive synergy) . Synergy was also demonstrated (P < .1) in the mouse 55 degrees C hot-plate test (i.p . route) and (P < .05) the rat Randall-Selitto yeast-induced inflammatory nociception model (i.v . and i.p . routes) . Critically, the enantiomers interacted less than synergistically in two side-effects of inhibition of colonic propulsive motility and impairment of rotarod performance . The racemate and the (+) enantiomer were active in a chronic (arthritic) inflammatory pain model . Taken together, these findings provide a rational explanation for the coexistence of dual components to tramadol-induced antinociception and might form the basis for understanding its clinical profile.

Hum Genet, 1993 Oct, 92(4), 405 - 9
A high-resolution cytogenetic map of human chromosome 12: localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization; Takahashi E et al.; We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding fluorescence in situ hybridization . The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands . This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers . Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes.

Food Chem Toxicol, 1993 Oct, 31(10), 739 - 44
In vitro toxicity evaluation of a product obtained from carmoisine using Tetrahymena pyriformis cells; Marathe SA et al.; The toxicity of carmoisine and of its metabolites after interaction with media components, was studied using the single cell system of Tetrahymena pyriformis . Carmoisine was not toxic to T . pyriformis . However, when autoclaved with media components such as bactopeptone or yeast extract, it gave rise to product(s) that completely inhibited the growth of T . pyriformis . Addition of glucose to the bactopeptone-carmoisine mixture before autoclaving significantly enhanced the formation of the toxic product . A similar effect was observed with other reducing sugars and with compounds containing sulfhydryl groups . The results indicate that carmoisine may undergo initial reduction, followed by a series of reactions leading to the formation of the toxic component(s).

Arch Pathol Lab Med, 1993 Oct, 117(10), 1043 - 6
Acanthamoeba infection presenting as skin lesions in patients with the acquired immunodeficiency syndrome; Tan B et al.; Acanthamoeba organisms are a well-known, although rare, cause of central nervous system infection in immunodeficient hosts, including those with the acquired immunodeficiency syndrome . Extracerebral acanthamebiasis, with the exception of contact lens-associated keratitis, is reported but little emphasized in the literature . We describe two patients with the acquired immunodeficiency syndrome in whom skin lesions were the primary manifestations of Acanthamoeba infection . Central nervous system disease was proved in one patient and suspected, but unproved, in the other . The skin lesions exhibited an intact epidermis with suppurative inflammation of the subcutis, associated with numerous amebic cysts and trophozoites . The amebic cyst walls stained with periodic acid-Schiff and Gomori's methenamine-silver stains, creating confusion with Blastomyces dermatitidis yeast in one instance . Immunofluorescence studies and culture identified the organisms as an Acanthamoeba species . Preliminary studies in one of the cases suggested a previously undescribed Acanthamoeba species as the etiologic agent . Our experience emphasizes that skin lesions may be the presenting sign of disseminated Acanthamoeba infection in patients with the acquired immunodeficiency syndrome.

Am J Hum Genet, 1993 Oct, 53(4), 864 - 73
Polymorphic microsatellites and Wilson disease (WD); Stewart EA et al.; Wilson disease (WD), an autosomal recessive disorder of copper metabolism, has been previously mapped to chromosome 13q . Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) at 13q14.3, as well as sequence-tagged sites for closely linked loci, are described . Two polymorphic microsatellite markers at D13S118 and D13S119 lie within 3 cM of WND . Two others (D13S227 and D13S228) were derived from a yeast artificial chromosome containing D13S31 . These were placed on a genetic linkage map of chromosome 13 and were typed in 74 multiplex WD families from a variety of geographic origins (166 affected members) . Multipoint analysis provides very high odds that the location of WND is between D13S31/D13S227/D13S228 and D13S59 . Previous odds with RFLP-based markers were only 7:1 more likely than any other location . Current odds are 5,000:1 . Preclinical testing of three cases of WD by using the highly informative polymorphic microsatellite markers is described . The markers described here ensure that 95% of predictive tests using DNA from both parents and from at least one affected sib will have an accuracy > 99%.

Diagn Microbiol Infect Dis, 1993 Oct, 17(3), 235 - 8
Aberrant Histoplasma capsulatum . Confirmation of identity by a chemiluminescence-labeled DNA probe; Sandin RL et al.; A cottony, light tan, filamentous fungus with pear-shaped microconidia and lacking tuberculated macroconidia was isolated from a bronchial lavage specimen . Subculture on several media at 37 degrees C failed to convert the fungus to a yeast form after several weeks; attempts at in vivo conversion in mice were also unsuccessful . Sera obtained several months apart showed M bands with Histoplasma capsulatum (HC) antigen by immunodiffusion and an increase in complement fixation titers with mycelial and yeast phase antigens of HC . Parallel identity was obtained on two occasions with exoantigen culture confirmation reagents for HC from Immuno-Mycologics as well as one of identity with Nolan reagents . Extracts from four Chrysosporium spp . strains had no identity reactions with HC with either kit . The fungus was identified as HC by the Accuprobe Histoplasma chemiluminescence-labeled DNA probe directed at ribosomal RNA, whereas all four Chrysosporium spp . isolates tested negative . DNA probes are a fast and accurate method to confirm the identity of aberrant fungal isolates.

Zhonghua Jie He He Hu Xi Za Zhi, 1993 Oct, 16(5), 278 - 80, 319
{Clinical investigation of erythrocyte function in patients with lung cancer}; Zhang YX et al.; With a yeast-erythrocyte-SPA rosette method, the function of erythrocyte immune adherence was determined in 49 cases of lung cancer, 20 cases of benign pulmonary diseases and 60 healthy persons . By means of simple morphologic method and pyrogallol autoxication method, the function of erythrocyte enhancing neutrophils phagocytosis and the activity of RBC-CuZnSOD were measured in some of these subjects . It was found that both the function of red cell immune adherence and the effect of erythrocyte enhancing phagocytosis in the patients with lung cancer were significantly weaker than in the cases of benign pulmonary diseases and the healthy subjects . The function of erythrocyte immune adherence tended to recover after effective surgical treatment . In comparison with the healthy persons, the activity of RBC-CuZnSOD was significantly lower in the patients with lung cancer . The mechanism and clinical significance of the changes in erythrocyte function were discussed.

Hum Mol Genet, 1993 Oct, 2(10), 1673 - 8
Fine mapping of the FSHD gene region orientates the rearranged fragment detected by the probe p13E-11; Wright TJ et al.; We have produced a fine restriction map around the locus D4F104S1 (previously designated D4S810); a probe to this locus, p13E-11, identifies a polymorphic EcoRI fragment containing 3.2kb tandem repeats and detects DNA rearrangements associated with facioscapulohumeral muscular dystrophy (FSHD) . We developed an STS (D4F106S1) which maps 2kb proximal to D4F104S1, and used this to isolate a 470kb YAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11) from the CEPH megabase library . Both YACs contain the loci D4S139, D4F35S1 and D4F104S1 . A cosmid library was produced from YAC y25C2E and two cosmid contigs constructed; a 115kb contig encompassing D4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extending distal to the EcoRI fragment detected by p13E-11 . A fine restriction map of both these contigs has been generated, allowing the orientation of the EcoRI fragment rearranged in FSHD to be determined . YAC y956A11 was used to confirm the integrity of y25C2E and the map of this region . 9B6A, a probe to the homeobox region of the tandem repeat D4Z4, identified a cross-hybridising sequence proximal to D4F104S1, however, p13E-11 does not detect this additional locus . CpG islands were identified between D4S139 and D4F35S1 and within each copy of the tandem repeat . The probe 9B6A detected each copy of the repeat motif, suggesting there is homeobox present in every copy of the 3.2kb repeat.

Genes Chromosomes Cancer, 1993 Oct, 8(2), 98 - 103
In situ hybridization to interphase nuclei in acute leukemia; Romana SP et al.; Numerical chromosome abnormalities were studied in 17 acute lymphoblastic leukemias and one hyperdiploid acute myeloblastic leukemia by fluorescence in situ hybridization (FISH) using YAC clones specific to chromosomes 21 and 6 . The results agreed well with cytogenetic findings . Hyperdiploid leukemias with more than 50 chromosomes usually had 4 copies of chromosome 21 and three of chromosome 6, while diploid and pseudodiploid cases were confirmed to have two copies of the two chromosomes . Interesting discrepancies were also observed . In one patient, trisomy 6 was detected by FISH but not by cytogenetics because of the probable inclusion of a chromosome 6 segment within a marker chromosome . The percentages of nuclei with 3 or 4 spots (chromosome 21) and three spots (chromosome 6) in hyperdiploid cells were significantly different in some patients, whereas they might be identical from cytogenetic data.

Nature, 1993 Sep 30, 365(6445), 459 - 62
Valosin-containing protein, VCP, is a ubiquitous clathrin-binding protein; Pleasure IT et al.; Clathrin is the structural protein of coated membranes involved in receptor-mediated endocytosis and aspects of Golgi sorting in eukaryotic cells . We have now detected a stoichiometric complex of clathrin with a novel protein of M(r) approximately 100,000 (100K) in lysates of different mammalian cells . Formation of the complex, which also includes the 70K heat-shock protein Hsc70, occurs within 15 min of synthesis . The 100K protein has been identified as valosin-containing protein (VCP; ref . 1), an early substrate for tyrosine phosphorylation on T-cell receptor activation . Further, VCP is the mammalian homologue of yeast Cdc48p (ref . 3) and is a member of a larger gene family that includes putative ATP-binding proteins involved in vesicle transport and fusion, 26S proteasome function, regulation of the expression of human immunodeficiency virus, and assembly of peroxisomes . The association with clathrin and the morphological and catalytic similarity to the chaperonin proteins indicate that VCP may modulate protein-protein interactions in membrane transport processes.

Gene, 1993 Sep 30, 132(1), 75 - 82
Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei; Affranchino JL et al.; Activation of the p34cdc2 protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs) . Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue . The amino acid sequence deduced for this gene (CYC1) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis . When expressed in fission yeast, CYC1 is able to rescue the defect of a temperature-sensitive cdc13 mutant, demonstrating that it is functional as a cell-cycle regulator . In trypanosome cells, CYC1 associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the complex displays histone H1 PK activity . Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYC1 accumulates as cells progress towards mitosis . These observations, taken together, suggest that CYC1 is a component of the active PK complex required for the control of trypanosome mitosis.

Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1394 - 400
Pre-messenger RNA splicing of transcripts synthesized from human small nuclear RNA gene promoters; White RA et al.; In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment . Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence . Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced . Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.

Philos Trans R Soc Lond B Biol Sci, 1993 Sep 29, 341(1298), 449 - 54
The Wellcome Lecture, 1992 . Cell cycle control; Nurse P; Genetic analysis using the fission yeast has provided a powerful methodology to investigate the eukaryotic cell cycle and its control . The onset of M-phase in fission yeast is controlled by a regulatory gene network which activates the p34cdc2 protein kinase encoded by the cdc2+ gene . The coupling of M-phase to the completion of S-phase also works through p34cdc2 . A similar network is operative in vertebrate cells . Future work will focus on the controls regulating onset of S-phase and on the mechanisms by which a cell duplicates itself in space during division.

Biochemistry, 1993 Sep 28, 32(38), 10057 - 66
Probing biomembrane interfacial potential and pH profiles with a new type of float-like fluorophores positioned at varying distance from the membrane surface; Kraayenhof R et al.; Fluorophores of a new type were synthesized to probe the electrostatic potential or pH profiles in the external interface of biomembranes . The probes consist of the pH-sensitive fluorophore 7-hydroxycoumarin, coupled to a tetradecyl (myristyl) tail by a spacer group of varying length . A positively charged group is included between the tetradecyl and spacer groups to encourage a float-like alignment in the membrane head-group region . Three probes of this type were compared with 4-heptadecyl-7-hydroxycoumarin the fluorophore of which is embedded in the lipid head-group domain . Thus, a ruler-type positioning of the fluorophores was obtained at about 0.2, 0.6, 1.0, and 1.3 nm from the surface . The membrane-bound probes were tested in well-defined liposomes prepared by extrusion with different surface charge densities and size . The predicted positioning of the float-like probes is supported by their binding behavior in liposomes and by steady-state and nanosecond time-resolved fluorescence anisotropy, as well as by their accessibility to different quenchers . The interfacial electrostatic potential (psi d) and pH (pHd) values were derived from the observed apparent pKa shifts of the probes . The obtained psi d and pHd profiles as function of the surface potential (psi 0) and distance from the membrane surface are in good harmony with predictions from nonlinear Gouy-Chapman theory . The electrokinetic potentials (zeta) of the liposome series, measured by Doppler-electrophoretic frequency shift of laser light scattering, are in good proportion to the probe data . When bound to yeast cells, these probes monitor interfacial changes in parallel with glucose-induced medium acidification.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1993 Sep 25, 21(19), 4524 - 9
HAPPY mapping of a YAC reveals alternative haplotypes in the human immunoglobulin VH locus; Walter G et al.; We have identified and sequenced 14 human immunoglobulin VH segments cloned in a yeast artificial chromosome, and have used a rapid PCR-based technique (HAPPY mapping, 12) to derive the order and approximate distances between them . The sequences mapped comprise thirteen germline VH segments and one rearranged VH3 gene . Comparison of our map with other data suggests the existence of at least two distinct haplotypes, differing in the presence or absence of the consecutive genes DP-78, DP-46 and DP-64, and in the duplication of segments DP-49 and DP-65 . Screening of ten individuals confirms the existence of both haplotypes, and indicates that both are common amongst the population.

Biochim Biophys Acta, 1993 Sep 23, 1174(3), 258 - 66
In vivo activity for initiation of DNA replication resides in a transcribed region of the human genome; Wu C et al.; The potential for autonomous replicating activity has been demonstrated, in the preceding paper, to exist in abundance in those sequences which are transcribed from the genome of human embryonic lung fibroblasts (IMR90) . In this paper we demonstrate for one sequence present in an 'O'-family homologous cDNA clone (clone 343), the likelihood of its in vivo activity by analysis of nascent replicated strands . Analysis of seven independent sources of human DNA with three different restriction enzyme digests failed to reveal any significant polymorphisms of the cDNA 343 homologous sequence . Homologous sequences were found in human, cow, mouse and monkey DNA, but not in rat, dog, rabbit, chicken or yeast DNA . An approx . 18 kbp fragment obtained as a clone from a genomic library of human embryonic lung fibroblasts was found to contain the sequence present in cDNA 343 in a 2.2 kbp EcoRI fragment . Sequence analysis of the clone provided three regions suitable for use as oligonucleotide primers in a PCR method of mapping the in vivo site of initiation of DNA replication . The initiation zone and a chromosomal origin for DNA replication are mapped to a region of approximately 1.6 kpb and were inclusive of the sequence detected in cDNA 343.

FEBS Lett, 1993 Sep 20, 330(3), 323 - 8
Isoprenylation of Rab proteins possessing a C-terminal CaaX motif; Joberty G et al.; Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion . The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways . They share distinct C-terminal cysteine motifs required for membrane association . Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins . This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro . Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate . Our study shows that the CaaX motif is required for isoprenylation.

Neurosci Lett, 1993 Sep 17, 160(1), 33 - 6
The c-fos gene and early-onset familial Alzheimer's disease; Bonnycastle LL et al.; A gene for early-onset familial Alzheimer's disease (FAD) is located on chromosome 14q24.3 . The c-fos gene (FOS) is also located in the same band of this chromosome and is thus a candidate for the FAD locus . A yeast artificial chromosome (YAC) clone was identified which contains FOS . This YAC also contains the short-tandem repeat polymorphic (STRP) locus D14S76, placing FOS in the FAD region between D14S53 and D14S43 . No recombinants were observed between D14S76 and FAD, and a maximum positive LOD score of 7.20 at a recombination fraction of 0.001 was observed for linkage of this marker to FAD . DNA sequence analysis was used to examine FOS in two affected subjects from an FAD family in which the chromosome 14 FAD locus is clearly responsible for the disease . The coding regions and parts of the 5' and 3' untranslated sequences of FOS were sequenced; no FAD-related mutations were observed . This work suggests that the FOS gene is not the chromosome 14 FAD locus although we cannot exclude the possibility that a mutation in an as yet unknown regulatory region is responsible for the disease . A new polymorphism was detected in the third intron of the gene.

Eur J Biochem, 1993 Sep 15, 216(3), 729 - 37
Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA . Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides; Haynes PA et al.; Prespore-specific antigen (PsA) is a cell-surface glycoprotein isolated from Dictyostelium discoideum, which is post-translationally modified by addition of carbohydrate to threonine residues of the carboxy-terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane . The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined . The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono-unsaturated fatty acid as the major component . The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol-specific phospholipase-C enzyme . The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 and Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 . The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide . The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of the D . discoideum GPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes . Compositional analysis of the carbohydrate attached to threonine residues in the carboxy-terminal peptide domain is also presented . The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8595 - 9
Giardiavirus double-stranded RNA genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift; Wang AL et al.; Giardiavirus is a small, nonenveloped virus comprising a monopartite double-stranded RNA genome, a major protein of 100 kDa, and a less abundant polypeptide of 190 kDa . It can be isolated from the culture supernatant of Giardia lamblia, a parasitic flagellate in human and other mammals, and efficiently infects other virus-free G . lamblia . A single-stranded copy of the viral RNA can be electroporated into uninfected G . lamblia cells to complete the viral replication cycle . Giardiavirus genomic cDNA of 6100 nt was constructed and its sequence revealed the presence of two large open reading frames that are separated by a -1 frameshift and share an overlap of 220 nt . The 3' open reading frame contains all consensus RNA-dependent RNA polymerase sequence motifs . A heptamer-pseudoknot structure similar to those found at ribosomal slippage sites in retroviruses and yeast killer virus was identified within this overlap . Immunostudies using antisera against synthesized peptides from four regions in the two open reading frames indicated that the 100- and 190-kDa viral proteins share a common domain in the amino-terminal region . But the 190-kDa protein makes a -1 switch of its reading frame beyond the presumed slippage heptamer and is therefore a -1 frameshift fusion protein similar to the gag-pol fusion protein found in retroviruses.

Cancer Res, 1993 Sep 15, 53(18), 4164 - 8
Loss of a p53-associated G1 checkpoint does not decrease cell survival following DNA damage; Slichenmyer WJ et al.; Cell cycle checkpoints regulate progression through the cell cycle . In yeast, loss of the G2 checkpoint by mutation of the rad9 gene results in increased genetic instability as well as increased sensitivity to ionizing radiation . In contrast, comparing clonogenic survival of cells which are isogeneic except for p53 functional status, we find that loss of a G1 checkpoint in mammalian cells is not associated with increased sensitivity to the lethal effects of ionizing radiation or a topoisomerase I inhibitor, camptothecin . These results indicate that increased sensitivity to DNA-damaging agents is not necessarily a defining feature of a mammalian cell cycle checkpoint . Furthermore, in light of a recent link of p53 function to radiation-induced apoptosis in hematopoietic cells, these observations suggest that p53-dependent apoptosis is a cell type-specific phenomenon and thus predict that the biological consequences of loss of p53 function will be cell type specific.

FEMS Microbiol Lett, 1993 Sep 15, 112(3), 329 - 33
Anti-Candida activity of four antifungal benzothiazoles; Bujdakova H et al.; Anti-Candida activity of 6-amino-2-n-pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV) . I and II exhibited good activity against the C . albicans yeast form, similar to IV . They were inhibitorily active against other Candida strains, IC50 values being of the order of 10(-5) M, which means better activity than IV . Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C . albicans strains, while II, III and IV were not active in these tests . III was the least active form of the compounds tested, IC50 values being of the order of 10(-4) M . All the compounds tested were highly active on a nystatin-resistant C . albicans mutant, with IC50s of the order of 10(-6) M-10(-5) M.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8509 - 13
Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint; Boldog FL et al.; The chromosome (p14.2;q24.1) translocation t(3;8) has been associated with hereditary renal cancer in one family . Based on cytogenetic analyses and loss-of-heterozygosity experiments, the 3p14 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development . The 3p14.2 region also contains FRA3B, the most sensitive fragile site induced by aphidicolin . A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was positioned between the t(3;8) breakpoint and an aphidicolin-induced 3p14 breakpoint . A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphidicolin-induced breakpoint on its telomeric side . A subsequent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint . A lambda sublibrary allowed isolation of clones spanning the rearrangement . Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library . We have identified a gene, referred to as HRCA1 (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint . On the basis of its chromosomal position, HRCA1 may be a candidate tumor suppressor gene.

J Immunol, 1993 Sep 15, 151(6), 3353 - 60
In vitro T cell immune responses to the preS2 antigen of the hepatitis B virus envelope protein in preS2 + S vaccine recipients . Absence of cross-reactivity of subtypes at a major T cell recognition site; Cupps TR et al.; The immune responses to hepatitis B virus envelope antigen were investigated in 16 vaccine recipients after immunization with a recombinant yeast-derived preS2 + S (adw) vaccine for hepatitis B virus . After the completion of the three-slot immunization series, all vaccine recipients developed antibody to the S domain and anti-preS2 antibody . In vitro proliferative responses to preS2 (120-174) peptide were demonstrated in 10 of 16 vaccine recipients . Although reactivity could be demonstrated through the length of the preS2 peptide, the principal site of proliferative activity was contained within the preS2 (146-165) region of the peptide . The principal T cell reactive site coincides with a region of significant amino acid variability of the different hepatitis B virus serotypes . Cross-reactivity with a serotype (ayw) not present in the preS2 + S vaccine could not be demonstrated at this widely recognized T cell epitope . The low level of cross-reactivity demonstrated in a limited subset of the vaccine recipients was mediated through nondominant T cell reactive sites contained in the relatively conserved preS2 (120-146) region of the molecule . The identification of widely recognized but serotype-specific T cell epitopes in the preS2 region of the hepatitis B virus envelope antigen may be an important consideration in future vaccine development.

J Biol Chem, 1993 Sep 15, 268(26), 19545 - 51
Novel ubiquitin-like ribosomal protein fusion genes from the nematodes Caenorhabditis elegans and Caenorhabditis briggsae; Jones D et al.; Among eukaryotes studied to date, homologs of the yeast 76-amino acid ribosomal protein have invariably been found to be cotranslated with ubiquitin . However, in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, a 70-amino acid domain with only 40% identity to ubiquitin is cotranslated with a homolog of the ribosomal protein . In the nematode ubiquitin-like (UbL) proteins, the nucleotide sequence of the UbL coding region is 92% identical in C . elegans and C . briggsae . The corresponding gene sequence contains a single intron at a location identical to that found in the polyubiquitin gene of C . elegans, further confirming that the ubl genes are evolutionarily related to ubiquitin . The ribosomal protein portion of the UbL polypeptide consists of 93 amino acids and is 68% identical to the human homolog . The ribosomal protein portion of UbL is longer than in other homologs, with the additional sequence being present as a basic carboxyl extension . The ubl gene is constitutively expressed in all life cycle stages of C . elegans . A comparison of the nematode UbL sequences with other ubiquitin-like genes reveals a pattern of sequence conservation, which suggests that the ubiquitin-like proteins may have conserved functional domains.

Nucleic Acids Res, 1993 Sep 11, 21(18), 4344 - 7
Cloning and characterization of the C . elegans histidyl-tRNA synthetase gene; Amaar YG et al.; In this paper, we report the cloning and sequencing of the C . elegans histidyl-tRNA synthetase gene . The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined . The gene size including flanking and coding regions is 2230 nucleotides long . Three small introns (45-50 bp long) are found to interrupt the open reading frame . The open reading frame translates to 523 amino acids . This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene . Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases . It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.

Nature, 1993 Sep 9, 365(6442), 185 - 8
Efficient catalysis of disulphide bond rearrangements by protein disulphide isomerase; Weissman JS et al.; Protein disulphide isomerase (PDI) is a highly abundant and ubiquitous eukaryotic protein that is essential for viability in yeast . Although PDI is thought to catalyse disulphide bond formation and isomerization during protein biosynthesis, PDI has been found previously to have only moderate effects (approximately 25-fold) on the rate of oxidative folding of proteins in vitro . In addition, PDI has been implicated in several apparently unrelated cellular functions . For example, PDI is the beta-subunit of prolyl 4-hydroxylase and is part of the triglyceride transfer complex . The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) is slow and inefficient in vitro . Here we report that PDI increases by a factor of 3,000-6,000 the rates of folding of kinetically trapped BPTI folding intermediates, in which native structure impedes disulphide bond formation . By contrast, PDI has only small effects on the rate of disulphide bond formation in intermediates that are oxidized readily in the absence of PDI . These results suggest that an important function of PDI is to catalyse disulphide bond formation and rearrangements within kinetically trapped, structured folding intermediates.

Nippon Rinsho, 1993 Sep, 51(9), 2359 - 63
{Molecular genetics of adrenoleukodystrophy}; Kaneko K; Human Xq28 region harbors many disease genes including genes for adrenoleukodystrophy, Emery-Dreifuss muscular dystrophy, X-linked centronuclear myopathy, and nephrotic diabetes inspidus . The genes for the diseases, however, have not been identified . On the other hand, only small number of transcribed sequences including G6PD gene, Gdx, P3, factor VIII gene, red and green color pigment genes, GABRA3 gene, L1 adhesion molecule gene, QM gene and so on have been identified at Xq28 . To identify the disease genes at Xq28 by positional cloning, it is essential to construct physical maps of the Xq28 region and to develop a strategy for identifying expressed genes . Macrorestriction maps of human Xq28 have been generated by pulsed field gel electrophoresis (PFGE) . With the recent development of yeast artificial chromosomes (YACs), major efforts have been focused on the generation of contigs of YACs from Xq28 . Recently, a putative ALD gene was identified . The gene named ALDP gene was partially deleted in 6 of 85 independent patients with ALD . In familial cases, the deletions segregated with the disease . The deduced protein sequence of ALDP shows significant sequence identity to a peroxisomal membrane protein of 70 K that is involved in peroxisome biogenesis and shares unexpected homology to ABC transporter gene.

Cancer Genet Cytogenet, 1993 Sep, 69(2), 100 - 7
Role of the gene on trisomic and pentasomic chromosome 13 in murine mammary tumorigenesis; Dofuku R et al.; To study a possible role(s) played by the trisomy and pentasomy of chromosome 13 in murine mammary tumors, we examined, in eight cloned established cell lines derived from a single BALB/c mammary tumor induced by MTV, a correlation between the presence of trisomy or pentasomy 13 and transformation parameters and in vivo tumorigenicity in syngeneic mice . We found that cell lines with a higher incidence of trisomy or pentasomy 13 in cells of diploid and tetraploid chromosome numbers, respectively, grew to a much higher cell density in flasks than did those with low incidence, and they formed tumors in syngeneic BALB/c mice, whereas those with a low incidence of trisomy or pentasomy 13 were poorly tumorigenic . The presence in the tumorigenic cells of trisomy or pentasomy 13 was not correlated with their growth in soft agar . Furthermore, other chromosomes manifested a wide range of copy numbers in the presence of trisomy or pentasomy 13, indicating that no chromosomes counteracted chromosome 13 to prevent the tumorigenicity . In light of the tumorigenic growth of the cells that maintain gene dosage of chromosome 13 at different ploidy levels, the possibility of the yeast G1 cyclin-like roles played by the gene(s) residing on chromosome 13 in murine mammary tumorigenesis is discussed.

J Cell Biol, 1993 Sep, 122(6), 1295 - 300
Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens; Evans E et al.; A simple micromechanical method has been used to directly measure the force of contraction in single mammalian phagocytes (blood granulocytes) during engulfment of large yeast pathogens . Both the time course of cell spreading over the yeast particle and increase in cell body contractile force were quantitated at three temperatures in the range of 23-35 degrees C . The surprising feature of the phagocyte response was that engulfment and cell body contraction occurred in a serial sequence: i.e., the phagocyte spread rapidly over the particle at a steady rate with no detectable cell body contraction; when spreading stopped, contraction force in the cell body then rose steadily to a plateau level that remained stationary until the next sequence of spreading and contraction . Both spreading and contraction exhibited abrupt start/stop kinetics . Also impressive, the cell contraction force stimulated by phagocytosis was quite large (approximately 10(-8) N)-two orders of magnitude larger than the force necessary to deform passive phagocytes to the same extent . If distributed uniformly over the cell cross section, the contraction force is equivalent to an average contractile stress of approximately 10(3) N/m2 (0.01 Atm) . These physical measurements in situ set critical requirements for the mechanism of force generation in granulocytes, imply that a major increase in network cross-linking accompanies build-up in contractile force and that subsequent network dissolution is necessary for locomotion.

Insect Biochem Mol Biol, 1993 Sep, 23(6), 691 - 701
Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta; Kramer KJ et al.; Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process . We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation . The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa . Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases . Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid . Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome . The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c . days 5-7 during the fifth larval instar . Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Hum Immunol, 1993 Sep, 38(1), 30 - 41
Differences in the central major histocompatibility complex between humans and chimpanzees . Implications for development of autoimmunity and acquired immune deficiency syndrome; Leelayuwat C et al.; Chimpanzees (Pan Troglodytes) and humans are closely related and belong to the same subfamily, Homininae . The approximately 1.8% genetic difference that exists between humans and the chimpanzees must be responsible for observed differences between these two species . It has been shown that chimpanzees can be infected with HIV, but AIDS has not been reported . Furthermore, the prevalence of autoimmune diseases may be low in this species . For instance, type II diabetes occurs, but type I (autoimmune) diabetes (IDDM), to our knowledge, has not been reported . In humans, susceptibility genes for MG and IDDM have been localized to the region between TNF and HLA-B . This region may also influence the rate of progression to death after HIV infection . We have identified differences in this region between humans and the chimpanzees . As shown by PFGE, the TNF to Patr-B region in the chimpanzees is approximately 130-160 kb shorter than the equivalent in humans . Southern and sequence analyses indicate that the deletions in chimpanzees (insertions in humans) include one copy of CL (approximately 10 kb) and the X sequences (< 30 kb) . Obviously, other deletions/insertions (approximately 120 kb) need to be identified . Since CL has been shown to be transcribed, the results imply the lack of the gene or, at least, a different gene copy number in the chimpanzees, and we propose that such differences may be relevant to the observed functional differences . We demonstrate here a strategy to identify critical genes responsible for disease development.

Hum Immunol, 1993 Sep, 38(1), 24 - 9
New major histocompatibility complex genes; Marshall B et al.; The MHC is a region of some 4 megabases that has been studied intensively owing to the large number of diseases that are associated with susceptibility genes within this region of the genome . The total number of genes located within the MHC is now approximately 100, but more can be predicted . Recently identified genes within the MHC include PERB6, a large gene producing multiple transcripts located between HLA-B and TNF, and PERB1, a member of the protein tyrosine kinase-gene family . PERB6 was identified by YAC probing of tissue blots, while PERB1 was identified by genomic sequencing.

Prenat Diagn, 1993 Sep, 13(9), 815 - 23
Rapid detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic mesenchymal cells; Bryndorf T et al.; We have devised and evaluated a rapid screening method for the detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic villus cells . We used non-radioactive in situ hybridization (ISH) with three chromosome-specific probes on overnight-attached mesenchymal cells from chorionic villi . A blind study was performed of 47 karyotypically normal samples, one triploid sample, two samples trisomic for chromosome 21, and two samples from a fetus with putative mosaicism (46/47, +21) . All samples were hybridized with the chromosome 18- and 21-specific probes . Thirty samples were additionally hybridized with the chromosome 13-specific probe . The test could be completed within 3-4 days of sampling . In samples disomic with respect to the probed chromosomes, an average of 2 per cent (range 0-9 per cent) had three hybridization signals . By contrast, in the samples trisomic for the probed chromosome(s), 57 per cent (chromosome 13), 51 per cent (chromosome 18), and an average of 74 per cent (55-86 per cent) (chromosome 21) of the nuclei exhibited three signals . In the putative mosaic samples, the number of nuclei with three chromosome 21-specific signals ranged from 41 to 69 per cent . We conclude that this technique rapidly and clearly distinguishes between normal and trisomic/triploid samples, and consequently may be of use in future prenatal diagnosis.

Biol Chem Hoppe Seyler, 1993 Sep, 374(9), 833 - 42
17th Fritz Lipmann Lecture . Wanderings (wonderings) in metabolism; Srere PA; Two of the most recent hypotheses concerning metabolism and its regulation are examined . First, the theory of distributive regulatory control of metabolic pathways is considered in light of experimental evidence concerning the lack of change in flux of a pathway when individual enzymes are either increased or decreased . In addition, it is known that when flux through pathways is either increased or decreased, one finds that the amount of all the enzymes of that path increase or decrease simultaneously . Second, the hypothesis of the existence of metabolons, complexes of sequential metabolic enzymes, is examined using the Krebs TCA cycle pathway as an example . The evidence from in vitro studies with pure enzymes to in vitro metabolic studies with yeast mutants is reviewed and evaluated . Unsolved problems concerning these hypotheses which still exist are considered.

Anticancer Res, 1993 Sep-Oct, 13(5C), 1763 - 7
Adoptive immunotherapy of advanced renal cell cancer using PHA-stimulated autologous lymphocytes; Morgan LR et al.; Patients with advanced renal cell carcinoma, previously failed maximal treatment with standard chemo-hormonal-radiation therapies, were treated with plant lectin phytohemagglutinin (PHA)-stimulated autologous peripheral blood lymphocytes in a 10-year study with a 16-year follow up period . In a phase I-II setting, 52 patients were given subcutaneously 40-80 x 10(6) PHA-stimulated lymphocytes weekly for 3 weeks and then escalated to a maximum number of 80 x 10(9) lymphocytes over the next 9 weeks at 3 week intervals . In vitro blastogenesis under study conditions (10 micrograms/ml PHA for 72 hr) measured by {3H}thymidine uptake was optimal with lymphocyte stimulating indexes approaching 300 . Lymphocytes obtained from patients with breast cancer, melanoma and renal cell carcinoma responded to PHA similarly to those from normal volunteers . All patients that responded developed erythematous reactions at the sites of injection; malaise, joint paint and chill-fever for 24-48 hr . The patients that responded the best were those with at least 1 positive reaction out of 4 skin tests (tuberculosis, yeast, dermatophytin, mumps) prior to therapy . All toxicity was transient and did not exceed Grade I based on criteria of the Southwest Oncology Group . The majority of patients developed a lymphopenia in the first 24 hr followed by a lymphocytosis 48-72 hr later . For some patients the lymphocytosis was as much as 30% atypical lymphocytes . Of 41 evaluable patients, there were 5 complete responses, 8 partial responses, 3 stable diseases, and 25 progressive disease . The overall response rate was 32% and the median survival was 2.8 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Microb Releases, 1993 Sep, 2(2), 73 - 9
Effect of temperature on survival of Legionella pneumophila in the aquatic environment; Paszko-Kolva C et al.; Although Legionella spp . are often isolated from natural aquatic habitats, outbreaks of legionellosis are rarely traced to these sources . To determine the fate of Legionella pneumophila in the environment, filtered and unfiltered river water and seawater microcosms, incubated at 4 degrees C and 26 degrees C, were inoculated with {3H}thymidine-labeled L . pneumophila cells . Survival in these microcosms was monitored using {3H}thymidine labeling and culture on buffered-charcoal yeast extract agar amended with alpha-ketoglutarate (BCYE alpha) . Immunofluorescent microscopy, direct fluorescent antibody staining, and acridine orange direct counts were also employed . To assess effects of grazing on Legionella, a duplicate set of samples was filtered through 2.0-microns Nuclepore filters to trap large protozoa . Over the test period, in the microcosms incubated at 4 degrees C, the culturable counts decreased ca . 1 log on BCYE alpha agar, with no substantial decline in thymidine count . Autoclaved seawater and river water controls held at 15 degrees C also showed no change in thymidine count . At 26 degrees C, a 3-log decline was observed in culturable counts, with ca . 1-log decline in thymidine counts . These results indicate that, although culturability declined by one to three orders of magnitude, when L . pneumophila microcosms were incubated at 4 degrees C and 26 degrees C, the cells remained metabolically active for extended periods, especially at 4 degrees C.

Keio J Med, 1993 Sep, 42(3), 91 - 4
Pityrosporum ovale and skin diseases; Faergemann J; Pityrosporum ovale is a lipophilic yeast belonging to the normal human cutaneous flora in adults . It is not only a saprophyte but also an opportunistic pathogen associated with: Pityriasis versicolor, Pityrosporum folliculitis, seborrhoeic dermatitis and some forms of atopic dermatitis . Even systemic infections have been described . In pityriasis versicolor P . ovale change from the blastospore to the mycelial form under the influence of predisposing factors such as high temperature, high relative humidity or endogenous factors such as greasy skin, sweating, heredity, immunosuppressive treatment or disorders . Topical treatment is often effective but short term treatment with fluconazole, ketoconazole or itraconazole is also effective . The great problem is recurrence and to avoid this a prophylactic treatment is mandatory . Pityrosporum folliculitis is a chronic disease characterized by pruritic follicular papules and pustules located primarily on the upper trunk, neck and upper arms . Under the influence of the same predisposing factors as in pityriasis versicolor P . ovale increase in numbers in the hair follicles . The main differential diagnosis is acne vulgaris . The effect of antifungal treatment is often dramatic . There are now many studies indicating that P . ovale plays an important role in seborrhoeic dermatitis . Many of these are treatment studies showing a good effect of antimycotics parallelled by a reduction in number of P . ovale . Severe seborrhoeic dermatitis often difficult to treat is associated with AIDS . In a recent study we have evidence for a slight T-cell defect in many patients with seborrhoeic dermatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

J Nat Prod, 1993 Sep, 56(9), 1500 - 5
Bioactive furanonaphthoquinones from Crescentia cujete; Hetzel CE et al.; Bioassay-directed fractionation of the MeCOEt extract of Crescentia cujete (Bignonaceae) resulted in the isolation of (2S,3S)-3-hydroxy-5,6-dimethoxydehydroiso-alpha-lapachone {1}, (2R)-5,6-dimethoxydehydroiso-alpha-lapachone {2}, (2R)-5-methoxydehydroiso-alpha-lapachone {3}, 2-(1-hydroxyethyl)naphtho{2,3-b}furan-4,9-dione {4}, 5-hydroxy-2-(1-hydroxyethyl)naphtho{2,3-b}furan-4,9-dione {5}, 2-isopropenylnaphtho{2,3-b}furan-4,9-dione {6}, and 5-hydroxydehydroiso-alpha-lapachone {7} . Compounds 1-3 are new, and all compounds are bioactive, showing selective activity towards DNA-repair-deficient yeast mutants . The isolation, structure elucidation, and biological activities of these compounds are reported.

EMBO J, 1993 Sep, 12(9), 3497 - 505
Protein phosphatase activity is required for light-inducible gene expression in maize; Sheen J; Chlorophyll accumulation and photosynthetic gene activation are two hallmarks of greening process in etiolated maize leaves in response to light signals . However, very little is known about the relevant signal transduction pathways mediating these essential processes that lead to photosynthetic competence . It is shown here that a potent and specific protein phosphatase 1 (PP1) and PP2A inhibitor, okadaic acid, efficiently blocks chlorophyll accumulation induced by light in etiolated maize leaves . In addition, the light-inducible expression of two photosynthetic fusion genes can be specifically suppressed by the structurally unrelated PP1 and PP2A inhibitors, okadaic acid and calyculin A, using a sensitive and physiological maize protoplast transient assay . The specificity and effective concentration of the inhibitors in vivo and in vitro strongly suggest that PP1 is required for transmitting light signals . Intriguingly, several partial cDNAs encoding novel as well as conserved PP1 can be identified in maize leaves using the polymerase chain reaction . Studies of chimeric promoters indicate that PP1 activity is essential for the interaction of multiple regulatory elements . Although PP1 and PP2A have been implicated in the suppression of gene activity in yeast and animals, the present data indicate that PP1 appears to be essential for light-dependent gene activation in plants.

J Clin Immunol, 1993 Sep, 13(5), 352 - 8
Cellular immune response of patients with uveitis to peptide M, a retinal S-antigen fragment; Nityanand S et al.; Peptide M, an 18-amino acid fragment from position 303 to position 320 of retinal S-antigen, produces experimental autoimmune uveitis (EAU), similar to that produced by native S-antigen, in several vertebrate species including nonhuman primates . It was observed that 12 of the 39 (30.7%) patients with uveitis, 1 of the 29 (3.4%) patients with systemic connective tissue disorders (CTD) without eye involvement, 2 of the 7 (5.8%) patients of CTD with uveitis, 1 of the 17 (5.8%) patients with diabetic retinopathy, and none of the 19 normal healthy controls showed a significant lymphoproliferative response to peptide M (stimulation index of 3 or more) . Yeast histone H3 peptide gave a positive response in 1 (2.5%), 2 (6.8%), 1 (14.2%), 2 (11.7%), and 2 (10.5%) individuals, respectively, in the different groups studied . In a few cases a positive response to yeast histone H3 peptide was observed without significant stimulation to peptide M . These findings indicate that peptide M could also be an immunogenic epitope of S-antigen in humans and be aetiopathologically related to uveitis in a subset of patients with this disease . However, unlike experimental animals, the responses to peptide M and yeast histone H3 were nonconcordant, necessitating further studies.

Genomics, 1993 Sep, 17(3), 744 - 7
A panel of sequence tagged sites for chromosome band 11q23; Tunnacliffe A et al.; A panel of sequence tagged sites (STSs) representing 30 markers previously assigned to human chromosome band 11q23 has been assembled . Eleven STSs represent cloned genes, and the remainder are from anonymous DNA segments . The STSs have been used in PCR experiments to localize their cognate sequences further with respect to five translocation breakpoints that define three intervals in 11q23 . Two of these translocation breakpoints have been mapped more precisely by the STS assignments . The STS panel will form a useful starting point for the generation of a genomic contig of band 11q23.

Genomics, 1993 Sep, 17(3), 682 - 93
Characterization of two chromosome 12 cosmid libraries and development of STSs from cosmids mapped by FISH; Montgomery KT et al.; We have constructed and characterized two related human chromosome 12-specific cosmid libraries . DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector . Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LL12NC01 and LL12NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats . One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH) . Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries . The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others . Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs) . These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12 . In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.

Genomics, 1993 Sep, 17(3), 676 - 81
Three human glutamate dehydrogenase genes (GLUD1, GLUDP2, and GLUDP3) are located on chromosome 10q, but are not closely physically linked; Deloukas P et al.; Yeast artificial chromosomes (YACs) of 340 and 370 kb that contain the functional human glutamate dehydrogenase gene (GLUD1) and the pseudogene GLUDP2, respectively, were isolated . These genes were not physically linked to each other nor to any other sequences homologous to the exons of GLUD1 . No additional GLUD sequences were found within at least 70 kb of the 5' and 175 kb of the 3' end of GLUD1 or 150 kb of either end of GLUDP2 . By in situ hybridization, GLUD1 was located at 10q23.3, GLUDP2 at 10q11.2, and another pseudogene of the GLUD gene family, GLUDP3, at 10q22.1 . DNA fragments of these three genes showed cross-hybridization to the loci assigned to the other two genes, but not to any other chromosomal locus . Thus, these three genes are located at distinct positions on chromosome 10q.

Genomics, 1993 Sep, 17(3), 651 - 6
Ordered shotgun sequencing, a strategy for integrated mapping and sequencing of YAC clones; Chen EY et al.; Ordered shotgun sequencing proposes to organize the mapping and sequencing of YACs with a hierarchical strategy that incorporates a feedback loop . Building on current protocols, a YAC is subcloned into plasmids, plasmid insert ends are sequenced, and the sequences are overlapped to create a partial map . Complete sequencing then starts with plasmids whose end-sequence tracts have overlapped, but to a minimal extent . The next plasmids to be sequenced are again selected for least overlap, striking out progressively to span the YAC with minimal directed gap-filling . Simulations support its feasibility and indicate that during the generation of the complete sequence, the approach facilitates the early choice of regions for selective sequencing, for example, for coding units . The sequencing of plasmids would also require less redundancy, and discriminate repetitive sequences more easily, than random sequencing across larger clones . The overall effort scales with YAC size and can be further reduced by additional mapping information.

Genomics, 1993 Sep, 17(3), 624 - 31
Multicolor FISH mapping with Alu-PCR-amplified YAC clone DNA determines the order of markers in the BRCA1 region on chromosome 17q12-q21; Flejter WL et al.; A gene designated BRCA1, implicated in the susceptibility to early-onset familial breast cancer, has recently been localized to chromosome 17q12-q21 . To date, the order of DNA markers mapped within this region has been based on genetic linkage analysis . We report the use of multicolor fluorescence in situ hybridization to establish a physically based map of five polymorphic DNA markers and 10 cloned genes spanning this region . Three cosmid clones and Alu-PCR-generated products derived from 12 yeast artificial chromosome clones representing each of these markers were used in two-color mapping experiments to determine an initial proximity of markers relative to each other on metaphase chromosomes . Interphase mapping was then employed to determine the order and orientation of closely spaced loci by direct visualization of fluorescent signals following hybridization of three probes, each detected in a different color . Statistical analysis of the combined data suggests that the order of markers in the BRCA1 region is cen-THRA1-TOP2-GAS-OF2-17HSD-248yg9-RNU 2-OF3-PPY/p131-EPB3-Mfd188- WNT3-HOX2-GP3A-tel . This map is consistent with that determined by radiation-reduced hybrid mapping and will facilitate positional cloning strategies in efforts to isolate and characterize the BRCA1 gene.

Genomics, 1993 Sep, 17(3), 575 - 81
Application of cDNA selection techniques to regions of the human MHC; Fan WF et al.; Identification of transcribed sequences by cDNA selection is a potentially rapid and efficient way of scanning large genomic DNA fragments for the presence of genes . To evaluate this approach further, we have applied it to three yeast artificial chromosomes (YACs) and examined the products obtained from a total of about 1100 kb from two regions of the human major histocompatibility complex (MHC) . One YAC was derived from an extensively studied portion of the Class II region of the MHC . The cDNAs recovered from this YAC included representatives of the previously described genes as well as one or more cDNA clones not described in the databases . A second YAC spanned about 330 kb of DNA surrounding the Class I gene HLA-A . In addition to Class I clones, 10 distinct cDNA products were identified from this YAC . A third YAC contained about 700 kb of human DNA, including 260 kb of overlap with the second YAC, and recovered an additional cDNA complementary to YAC B30 H3 DNA . Overall, the method is shown to be able to detect very scarce cDNAs and to detect a large fraction of coding sequences in YAC clones . Advantages and limitations of the approach are discussed.

Hum Mol Genet, 1993 Sep, 2(9), 1389 - 96
Physical and transcriptional mapping of DXS56-PGK1 1 Mb region: identification of three new transcripts; Gecz J et al.; Several new techniques for isolation expressed sequences have been recently described considerably speeding up the identification of unknown genes . Here we present a transcriptional map of the 1 Mb DXS56-PGK1 region in Xq13.3 . Rare cutter restriction site mapping, direct cDNA selection on membrane discs and probing of Northern blots with total YAC DNA, were the methods explored in order to achieve this goal . In addition to three known genes from this region which have been recloned, two new cDNA clones corresponding to two new genes were isolated, mapped and characterized . Moreover one more transcript, highly expressed in placenta, has been detected in the region with a total YAC as a probe . In summary there are at least six genes known to reside in the DXS56-PGK1 region . As several human disease gene loci (i.e . SCID, CMTX1, WWS, MRX, XDP, ASB) were tightly linked to the markers from the region (PGK, CA repeats), the three new transcripts may be considered as their potential candidate genes.

Hum Mol Genet, 1993 Sep, 2(9), 1361 - 8
Cloning of a novel, anonymous gene from a megabase-range YAC and cosmid contig in the neurofibromatosis type 2/meningioma region on human chromosome 22q12; Xie YG et al.; In order to permit detailed characterization of meningioma cases showing deletions within chromosomal band 22q12 and further systematically clone genes located within this region, we established a genomic YAC and cosmid contig which encompasses a region in excess of 1000 kb of 22q12 . The YAC contig consists of 6 YAC clones arranged into 5 overlapping steps covering more than 1100 kb . Two corresponding cosmid contigs consisting of 40 steps of overlapping groups of cosmids encompasses 900-1000 kb . This set of genomic clones provides a detailed physical map of this part of chromosome 22 and constitutes a basis for the isolation and characterization of genes that may be located within this chromosomal region . Employing the exon-amplification method on two cosmids from the contig, we cloned a novel, anonymous gene, pK1.3, which potentially encodes a protein of 683 amino acids with a predicted molecular weight of of 78.5 kD . Its 2.7 kb mRNA is expressed ubiquitously . We estimated the genomic size of this gene to 100-150 kb, and it is located in the immediate centromeric vicinity of the neurofibromatosis 2 (NF2) tumor suppressor gene.

Bioessays, 1993 Sep, 15(9), 595 - 603
The spliceosome; Lamond AI; The spliceosome is a large RNA-protein complex that catalyses the removal of introns from nuclear pre-mRNA . A wide range of biochemical and genetical studies shows that the spliceosome comprises three major RNA-protein subunits, the U1, U2 and {U4/U6.U5} small nuclear ribonucleoprotein particles (snRNPs), and an additional group of non-snRNP protein splicing factors . Rapid progress is being made in unravelling the interactions which take place between these factors during the splicing reaction . The emerging picture of the spliceosome reveals a highly dynamic structure that assembles on pre-mRNA transcripts in a stepwise pathway and is organised, at least in part, by complex RNA base-pairing interactions between the small nuclear RNAs (snRNAs) and the intron substrate . Many of these interactions can be detected both in mammalian and yeast spliceosomes, suggesting that the basic splicing mechanism is an ancient one that has been highly conserved during evolution.

Naunyn Schmiedebergs Arch Pharmacol, 1993 Sep, 348(3), 332 - 7
Identification of P450 enzymes involved in metabolism of verapamil in humans; Kroemer HK et al.; The calcium channel blocker verapamil{2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6- azaoctanitrile} is widely used in the treatment of hypertension, angina pectoris and cardiac arrhythmias . The drug undergoes extensive and variable hepatic metabolism in man with the major metabolic steps comprising formation of D-617 {2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile} and norverapamil {2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile} . The enzymes involved in metabolism of verapamil have not been characterized so far . Identification of these enzymes would enable estimation of both interindividual variability in verapamil metabolism introduced by the respective pathway and potential for metabolic interactions . We therefore characterized the enzymes involved in formation of D-617 and norverapamil . The maximum rate of formation of D-617 and norverapamil was determined in the microsomal fraction of 21 human livers which had been previously characterized for the individual expression of various P450 enzymes (CYP1A2, CYP2C, CYP2D6, CYP2E1 and CYP3A3/4) by means of Western blotting . Specific antibodies directed against CYP3A were used to inhibit formation of D-617 and norverapamil . Finally, formation of both metabolites was investigated in microsomes obtained from yeast cells which were genetically engineered for stable expression of human P450 . Formation of D-617 was correlated with the expression of CYP3A (r = 0.85; P < 0.001) and CYP1A2 (r = 0.57; P < 0.01) in the microsomal fraction of 21 human livers after incubation with racemic verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)

J Med Virol, 1993 Sep, 41(1), 30 - 4
Immunogenicity of 20 micrograms of recombinant DNA hepatitis B vaccine in healthy neonates: a comparison of three different vaccination schemes; del Canho R et al.; The immunogenicity of a full dose (20 micrograms) of recombinant DNA yeast-derived hepatitis B vaccine (Engerix-B) was assessed in healthy neonates in order to compare three candidate vaccination schemes . After randomization 162 newborns of hepatitis B surface antigen (HBsAg) negative mothers entered the study . Neonates received hepatitis B vaccine according to a four-dose vaccination scheme starting either at month 3 (scheme I: months 3, 4, 5, and 11) or at birth (scheme III: months 0, 1, 2, and 11) . Another group of neonates received hepatitis B vaccine according to a three-dose scheme starting at birth (scheme II: months 0, 1, and 6) . Serious adverse reactions were not observed; 2.5% of the vaccinated newborns suffered mild transient local symptoms . The vaccine was highly immunogenic irrespective of vaccination scheme; all infants developed anti-HBs levels > or = 10 IU/L, 97% > or = 100 IU/L . The immunogenicity of hepatitis B vaccine after primary and booster vaccinations, administered in the four-dose scheme started at birth, was significantly higher (P < 0.05) than in the three-dose scheme started at birth . Hepatitis B vaccination according to the four-dose scheme started at month 3 produced significantly higher (P < 0.05) antibody levels in comparison to the four-dose scheme started directly after birth . This study showed that a four-dose hepatitis B vaccination scheme starting at month 3 resulted in the highest antibody levels of the three schemes investigated and can be recommended for incorporation in the Expanded Programme on Immunization in The Netherlands.

Nat Genet, 1993 Sep, 5(1), 22 - 30
Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice {corrected}; Lamb BT et al.; Overexpression of the gene encoding the beta-amyloid precursor protein (APP) may have a key role in the pathogenesis of both Alzheimer's disease (AD) and Down Syndrome (DS) . We have therefore introduced a 650 kilobase (kb) yeast artificial chromosome (YAC) that contains the entire, unrearranged 400 kb human APP gene into mouse embryonic stem (ES) cells by lipid-mediated transfection . ES lines were generated that contain a stably integrated, unrearranged human APP gene . Moreover, we demonstrate germ line transmission of the APP YAC in transgenic mice and expression of human APP mRNA and protein at levels comparable to endogenous APP . This transgenic strategy may prove invaluable for the development of mouse models for AD and DS.

Plant Physiol, 1993 Sep, 103(1), 205 - 12
Uncoating of clathrin-coated vesicles by uncoating ATPase from developing peas; Kirsch T et al.; A cytosolic ATPase (an enzyme that dissociates clathrin from clathrin-coated vesicles in the presence of ATP) was isolated from developing pea (Pisum sativum L.) cotyledons using chromatography on ATP-agarose . After chromatography on phenyl Sepharose, the fraction with uncoating activity was enriched in a doublet of 70-kD peptides . Using chromatofocusing, it was possible to produce fractions enriched in the upper component of the doublet of 70-kD peptides; these fractions still retained ATP-dependent uncoating activity . In western blot analysis, antibodies against a member of the 70-kD family of heat-shock proteins interacted with the upper component of the doublet of the 70-kD peptides from the phenyl Sepharose-purified fractions . On the basis of these data, it appears that the uncoating ATPase may be a member of the 70-kD family of heat-shock proteins . The uncoating activity removed clathrin from both pea and bovine brain clathrin-coated vesicles . The uncoating ATPase from bovine brain also uncoated coated vesicles from peas . Pea clathrin-coated vesicles that were prepared by three different methods were uncoated to different extents by the plant uncoating ATPase . Different populations of clathrin-coated vesicles from the same preparation showed differential sensitivity to the uncoating ATPase . Limited proteolysis of the clathrin light chains in the protein coat abolished the susceptibility of the clathrin-coated vesicles to the uncoating ATPase . The properties of the uncoating ATPase isolated from developing pea cotyledons are similar to those of uncoating ATPases previously described from mammalian and yeast systems . It appears that despite dissimilarities in composition of the clathrin components of the vesicles from the respective sources, uncoating is achieved by a common mechanism.

Ukr Biokhim Zh, 1993 Sep-Oct, 65(5), 69 - 76
{Interaction of immobilized lectin from Leucojum vernum L . with polysaccharides and glycoproteins}; Antoniuk LIa et al.; Interaction of D-mannose-specific lectin from Leucojum vernum L . with polysaccharides and glycoproteins has been studied on the column of immobilized lectin and by precipitation . Lectin is found to precipitate human and bovine thyroglobulin, yeast mannan, mannofucogalactans from Aphyllophores mushrooms and rystomycin sulfate . Lectin does not interact with yeast glycogen and gel sephadex and this differentiates it from Leucojum vernum L., Concanavalin A and pea lectin . All glycoconjugates are divided into three groups: those which are not bound with immobilized lectin; partly bound with Leucojum vernum lectin and completely bound with the column . Glycoconjugates that are partly bound with immobilized lectin are, probably, glycosyl moiety . The obtained results are discussed.

Mamm Genome, 1993 Sep, 4(9), 523 - 30
Mapping the murine Xce locus with (CA)n repeats; Simmler MC et al.; The X Chromosome (Chr) controlling element locus (Xce) in the mouse has been shown to influence the X inactivation process . Xce maps to the central region of the X Chr, which also contains the Xist sequence, itself possibly implicated in the X inactivation process . Three microsatellite markers spanning the Xist locus have been isolated from an Xist containing YAC . All three microsatellite markers showed complete linkage with Xce in recombinants for the central span of the mouse X Chr between Ta and Moblo and strong linkage disequilibrium with Xce in all but one of the inbred mouse strains tested . In the standard Xceb typing strain JU/Ct, the two microsatellites most closely flanking Xist fail to carry the allelic forms expected if Xist and Xce are synonymous . Alternative explanations for this finding are presented in the context of our search for understanding the relation between Xist and Xce.

Semin Dermatol, 1993 Sep, 12(3), 255 - 65
The molecular genetics of incontinentia pigmenti; Gorski JL et al.; Incontinentia pigmenti (IP) is an unusual and fascinating disorder of the developing neuroectoderm . IP is an X-linked dominant disease characterized by congenital and age-related dermatologic abnormalities and significant neurological, ophthalmologic, and dental anomalies . Two distinct IP gene loci, IP1, mapped to Xp11.21, and IP2, mapped to Xq28, have been identified . The necessary prerequisites for cloning the IP1 gene by a positional cloning approach are available . Ten DNA markers have been mapped to a region between IP1 X-chromosomal translocation breakpoints within region Xp11.21 . Approximately 60% of the 2,500-kb region between IP1 X-chromosomal translocation breakpoints has been cloned in yeast artificial chromosome clones.

Semin Dermatol, 1993 Sep, 12(3), 247 - 54
The Wiskott-Aldrich syndrome; Peacocke M et al.; Skin diseases manifesting classic sex-linked recessive patterns of inheritance provide straightforward problems in the mapping of disease loci . In contrast to autosomal disorders, in which the abnormal gene might be found on any chromosome, sex-linked diseases are found only on the human X chromosome . Thus, with a reasonable number of polymorphic DNA probes and families with living affected and unaffected males, disease loci can be easily mapped to the relevant subregions of the X chromosome . The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by eczema, immunodeficiency, and thrombocytopenia . Boys with WAS suffer from skin diseases, bleeding problems, recurrent infections, and lymphoid malignancies . In contrast, females who carry the WAS gene are entirely normal, as the abnormal X chromosome is selectively inactivated in cells of hematopoietic origin . Using restriction fragment length polymorphisms (RFLPs) and appropriate families, the WAS locus has been mapped to the proximal portion of the short arm of the X chromosome (Xp11) . Prenatal diagnosis is now possible using specific RFLP markers . Moreover, combining RFLP studies with methylation analysis has allowed identification of all female carriers . Although the abnormal gene and protein that are responsible for WAS are currently unknown, studies using yeast artificial chromosomes containing portions of human Xp11 should ultimately allow for the cloning and characterization of the WAS gene . As this gene is expressed primarily in cells of bone marrow origin, WAS is an excellent candidate disease for gene therapy.

Semin Respir Infect, 1993 Sep, 8(3), 183 - 90
Bone-marrow transplantation and related infections; Crawford SW; The utility of bone-marrow transplantation (BMT) in the treatment of malignant and hematopoietic diseases is increasing . Additionally, refinements in the use of peripheral stem cells, unrelated marrow donors, and management of immunological complications of graft-versus-host reactions have contributed to the increase in the number of BMT performed each year . At the same time, the spectrum of infections associated with BMT are evolving . Improved prophylactic measures are decreasing the severity and frequency of bacterial, yeast and cytomegalovirus and Herpes simplex viral infections . However, other viruses are now emerging as pathogens . Common community-acquired respiratory viruses have caused significant morbidity and reports of human herpes virus-6 (HHV-6) infections in diffuse pneumonia raise the specter of additional previously unrecognized viral pathogens . Most alarming however, is the apparent increase in infections caused by filamentous fungi, which appear to develop despite anti-fungal prophylaxis . Improvement in preventive and treatment options for fungal disease is crucial to continued improvement in the outcome of BMT recipients.

J Biochem (Tokyo), 1993 Sep, 114(3), 432 - 7
Purification of an AU-rich RNA binding protein from Sarcophaga peregrina (flesh fly) and its identification as a Thiolase; Nanbu R et al.; A protein that binds to the AU-rich sequence in the 3'-untranslated region of sarcotoxin IIA mRNA was purified from a Sarcophaga pupal extract to near homogeneity . The molecular mass of this protein was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis . The partial amino acid sequences of two peptides obtained from the 39 kDa protein showed striking similarities to partial amino acid sequences of rat and yeast 3-oxoacyl-CoA thiolase, suggesting that this protein is a Sarcophaga thiolase . In fact, the purified 39 kDa protein was found to have thiolase activity . Moreover, rat mitochondrial 3-oxoacyl-CoA thiolase showed affinity to the AU-rich RNA . These suggest that the RNA binding activity is an intrinsic character of thiolase.

Chromosoma, 1993 Sep, 102(8), 563 - 74
Two early replicated, developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis; Diller JD et al.; The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells . We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase . A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin . In contrast, the LAV1-2 gene appears to be surrounded by several origins . Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used . Replication forks collide at preferred sites within this domain . These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase . Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process . Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.

Hepatology, 1993 Sep, 18(3), 497 - 502
Antibody response to core, envelope and nonstructural hepatitis C virus antigens: comparison of immunocompetent and immunosuppressed patients; Lok AS et al.; Some immunosuppressed patients with hepatitis C virus infection do not have detectable levels of antibody to hepatitis C virus on second-generation enzyme immunoassay . Antibodies to the envelope and nonstructural region 5 proteins have not been examined . Four groups of patients with hepatitis C virus infection were studied: (a) 20 immunocompetent patients, (b) 15 hemodialysis patients, (c) 17 kidney transplant recipients and (d) 3 acute leukemia patients who underwent bone marrow transplantation . Serum samples were tested for antibody to hepatitis C virus with a second-generation enzyme immunoassay and multi-antigen enzyme immunoassays and for hepatitis C virus RNA with a nested polymerase chain reaction assay . All the immunocompetent patients reacted to C25, C22 and C33C; 90% reacted to nonstructural region 5 antigen and 80% reacted to C100-3 . Only 55% reacted against yeast-derived e1 and e2 antigens, but all reacted against vaccinia virus--expressed N e1 and e2 antigens, indicating that the envelope epitopes are conformational and glycosylated . Sixty-five percent to 90% of dialysis and kidney transplant patients reacted to C25, C22 and N e1 and e2, but only 12% to 60% reacted to C100-3, C33C and nonstructural region 5 antigen . Diminution or loss of reactivity to hepatitis C virus antigens was observed after kidney and bone marrow transplantation, with C25 and N e1 and e2 less affected . Our data suggest that incorporation of C25 and N e1 and e2 antigens in the assay for antibody to hepatitis C virus would improve the detection of hepatitis C virus infection in immunosuppressed patients.

EMBO J, 1993 Sep, 12(9), 3427 - 36
Two distinct mechanisms for negative regulation of the Wee1 protein kinase; Tang Z et al.; The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein . To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts . We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle . The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis . The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase . This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase . These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein . Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.

Biochem Biophys Res Commun, 1993 Aug 31, 195(1), 393 - 9
Cloning of a cDNA which encodes a novel ubiquitin-like protein; Kumar S et al.; The cloning of a mouse cDNA, Nedd-8, which encodes a small novel protein of 81 amino acid residues with a relative molecular mass of 9 kDa, is described . The putative Nedd-8 product is approximately 60% identical to ubiquitin protein . The 0.6 kb mRNA for Nedd-8 gene can be detected in various mouse tissues and cell lines derived from various sources . Nedd-8 probe hybridizes to genomic DNA from various vertebrate species as well as yeast, indicating high degree of interspecies conservation . These data suggest that like ubiquitin, the product of this gene may play some essential role in eukaryotic cellular metabolism.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4039 - 45
Non-universal decoding of the leucine codon CUG in several Candida species; Ohama T et al.; It has been reported that CUG, a universal leucine codon, is read as serine in an asporogenic yeast, Candida cylindracea . The distribution of this non-universal genetic code in various yeast species was studied using an in vitro translation assay system with a synthetic messenger RNA containing CUG codons in-frame . It was found that CUG is used as a serine codon in six out of the fourteen species examined, while it is used for leucine in the remaining eight . The tRNA species responsible for the translation of codon CUG as serine was detected in all the six species in which CUG is translated as serine . The grouping according to the CUG codon assignments in these yeast species shows a good correlation with physiological classification by the chain lengths of the isoprenoid moiety of ubiquinone and the cell-wall sugar contained in the yeasts . The six Candida species examined in which CUG is used as serine belong to one distinct group in Hemiascomycetes.

Nucleic Acids Res, 1993 Aug 25, 21(17), 3997 - 4004
A general protocol for evaluating the specific effects of DNA replication inhibitors; Levenson V et al.; Inhibitors of DNA replication in mammalian cells are of great interest because of their potential use in chemotherapy and in cell synchronizing protocols in the laboratory . We have used a combination of isotopic labelling protocols and a two-dimensional gel replicon mapping procedure to determine the specific effects of five different replication inhibitors in cultured cells . Utilizing this protocol, we show that hydroxyurea, aphidicolin, and cytosine arabinoside, three known chain elongation inhibitors, are rather ineffective at preventing fork progression even at relatively high concentrations . In contrast, two related compounds that have been suggested to be G1/S inhibitors (mimosine and ciclopyrox olamine {CPX}) actually appear to inhibit initiation at origins . One of these agents (CPX) appears also to inhibit replication in yeast, opening the possibility that the gene encoding the target (initiator?) protein can first be identified in yeast by genetic approaches and can then be used to isolate the mammalian homologue.

Science, 1993 Aug 20, 261(5124), 1041 - 4
Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia; Liu P et al.; The pericentric inversion of chromosome 16 {inv(16)(p13q22)} is a characteristic karyotypic abnormality associated with acute myeloid leukemia, most commonly of the M4Eo subtype . The 16p and 16q breakpoints were pinpointed by yeast artificial chromosome and cosmid cloning, and the two genes involved in this inversion were identified . On 16q the inversion occurred near the end of the coding region for CBF beta, also known as PEBP2 beta, a subunit of a heterodimeric transcription factor regulating genes expressed in T cells; on 16p a smooth muscle myosin heavy chain (SMMHC) gene (MYH11) was interrupted . In six of six inv(16) patient samples tested, an in-frame fusion messenger RNA was demonstrated that connected the first 165 amino acids of CBF beta with the tail region of SMMHC . The repeated coiled coil of SMMHC may result in dimerization of the CBF beta fusion protein, which in turn would lead to alterations in transcriptional regulation and contribute to leukemic transformation.

Biochemistry, 1993 Aug 17, 32(32), 8083 - 91
Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation; Van Doren SR et al.; Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides have been constructed . The substitution of aspartate for glycine at position 133 in the Rb . sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolated and characterized in yeast by Gatti et al . {Gatti, D.L., Meinhardt, S.W., Ohnishi, T., & Tzagoloff, A . (1989) J . Mol . Biol . 205, 421-435}, allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinol:cytochrome c2 oxidoreductase on flash activation of the photosynthetic chain . The impaired catalysis in this mutant complex is localized to the quinol oxidizing site . The apparent second-order rate constant for reduction of cytochrome bH via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with its apparent activation barrier being increased relative to that of the wild-type . Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact . Reversion properties of these strains are consistent with the mutated residues being essential.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1207 - 15
Mapping a putative pyruvoyl decarboxylase active site to human cytomegalovirus open reading frame UL77; Yoakum GH; Human cytomegalovirus is present as an apparently innocuous infection among a large proportion of the adult population that causes serious disease when congenitally transferred (1) . Human cytomegalovirus disease may become life threatening when found as an infection of immunocompromised transplant and AIDS patients (1,2) . We now map a putative pyruvoyl decarboxylase enzyme prosthetic group, known to be essential to the active site of this class of enzymes, to Human cytomegalovirus open reading frame UL77 . The significance of finding a putative pyruvoyl decarboxylase enzyme prosthetic group among human cytomegalovirus open reading frame codons is p = 0.001 . The UL77 polypeptide was aligned with human, yeast and bacterial S-AdoMet decarboxylase enzymes . Alignment of UL77 and S-AdoMet decarboxylase enzymes indicates that UL77 may contain a similar activity.

J Biol Chem, 1993 Aug 15, 268(23), 17205 - 10
Molecular cloning and characterization of cDNAs for the gamma- and epsilon-subunits of mitochondrial F1F0 ATP synthase from the sweet potato; Morikami A et al.; Mitochondrial F1F0 ATP synthases purified from dicotyledonous plants contain six different subunits named alpha, beta, gamma, delta, delta', and epsilon . Our previous N-terminal amino acid sequence analyses indicated that the gamma- and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma- and epsilon-subunits of animal mitochondrial F1, respectively (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T . (1989) J . Biol . Chem . 264, 3183-3186) . A cDNA clone for the gamma-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide hybridization selection of a cDNA library, and a cDNA clone for the epsilon-subunit was isolated by reverse polymerase chain reaction and hybridization selection of a cDNA library by the polymerase chain reaction product . The 1.4-kilobase long cDNA for the gamma-subunit contained a 978-base pair open reading frame coding for a precursor for the gamma-subunit . The mature gamma-subunit is composed of 281 amino acids, and its sequence showed significantly higher similarities with the gamma-subunit of animal mitochondrial F1 and bacterial F1 compared with the gamma-subunit of chloroplast CF1 from plants . The precursor for the gamma-subunit contained N-terminal presequence of 45 amino acid residues . By contrast, the 0.46-kilobase long cDNA for the epsilon-subunit contained a coding sequence of 207-base pairs for the mature epsilon-subunit of 69 amino acid residues that is preceded by an ATG codon suggesting that the epsilon-subunit is synthesized without the cleavable presequence for mitochondrial import . The amino acid sequence of the epsilon-subunit of sweet potato mitochondrial F1 showed similarities of 25 and 36% amino acid positional identity with the epsilon-subunits of mitochondrial F1 from yeast and bovine, respectively.

Cell, 1993 Aug 13, 74(3), 463 - 74
Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase; Heald R et al.; The wee1 tyrosine kinase and cdc25 tyrosine phosphatase of fission yeast play antagonistic roles in the induction of mitosis through cdc2 regulation . We show here that the human wee1-like tyrosine kinase is a nuclear protein that ensures the completion of DNA replication prior to mitosis in cells expressing otherwise catastrophic levels of cdc2 activators . Paradoxically, wee1-rescued cells display very high levels of mitotic cdc2 kinase activity . We account for this anomaly by our observation that the cdc2 activator, cdc25C, is a cytoplasmic protein that, like cyclin B1, enters the nucleus at the G2/M transition . Thus, cdc2 is likely to be activated in the cytoplasm and requires nuclear localization to initiate both cytoplasmic and nuclear mitotic transformations . The human wee1 kinase appears to coordinate the transition between DNA replication and mitosis by protecting the nucleus from this cytoplasmically activated cdc2 kinase.

J Biol Chem, 1993 Aug 5, 268(22), 16717 - 24
The role of conserved amino acids in substrate binding and discrimination by photolyase; Baer ME et al.; DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA . We have utilized chemical modification and site-directed mutagenesis to probe the interactions involved in substrate recognition by the yeast photolyase Phr1 . Lys517 was protected from reductive methylation in the presence of substrate, but not in its absence, and the specific and nonspecific association constants for substrate binding by Phr1 (Lys517-->Ala) were decreased 10-fold . These results establish a role for Lys517 in substrate binding . Mutations at Arg507, Lys463, and Trp387 reduced both the overall affinity for substrate and substrate discrimination . Sites of altered interactions in ES complexes were identified by methylation and ethylation interference techniques . Interaction with the base immediately 3' to the dimer was altered in the Phr1(Lys517-->Ala) . DNA complex, whereas interactions with the phosphate and base immediately 5' to the dimer were reduced when Phr1(Arg507-->Ala) bound substrate . Multiple interactions 5' and 3' to the dimer were perturbed in complexes containing Phr1(Trp387-->Ala) or Phr1(Lys463-->Ala) . In addition the quantum yield for dimer photolysis by Phr1(Trp387-->Ala) was reduced 3-fold . The locations of these mutations establish that a portion of the DNA binding domain is comprised of residues in the highly conserved carboxyl-terminal half of the enzyme.

J Biol Chem, 1993 Aug 5, 268(22), 16655 - 63
Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase . A specific biochemical and cytological marker for a unique membrane fraction in rat liver; Cui Z et al.; Phosphatidylethanolamine N-methyltransferase catalyzes the synthesis of phosphatidylcholine from phosphatidylethanolamine and is most active in liver . A cDNA for this enzyme from a rat liver cDNA library has been cloned, sequenced, and expressed in COS-1 cells, McArdle-RH7777 rat hepatoma cells, and Sf9 insect cells . The expressed protein was capable of converting phosphatidylethanolamine into phosphatidylcholine in intact COS-1 cells, which normally have very low methyltransferase activity . The calculated molecular mass of the methyltransferase protein is 22.3 kDa, which is equivalent to that of the pure protein isolated from rat liver . Comparison of the sequence of the cloned rat liver methyltransferase with the yeast phosphatidylethanolamine methyltransferase PEM2 gene product revealed 44% identical amino acids and 68% similarity in the two predicted protein sequences . A polyclonal antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal region of the enzyme and was affinity purified . The antibody recognized a single protein with a molecular mass of approximately 20 kDa when either rat liver proteins or proteins derived from the transfected COS-1 cells were electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate . Surprisingly, the antibody exhibited no reactivity with endoplasmic reticulum proteins, even though the major phosphatidylethanolamine methyltransferase activity resides on this subcellular organelle . Instead, the antibody specifically recognized a protein in a unique subcellular membrane fraction purified from a crude mitochondrial preparation on a Percoll gradient . Immunocytochemical examination by electron microscopy showed positive labeling only in unique regions of the hepatocytes . The data suggest that this phosphatidylethanolamine methyltransferase is a specific marker for this unique membrane fraction.

J Biol Chem, 1993 Aug 5, 268(22), 16631 - 8
Sequencing and cloning of human prolylcarboxypeptidase (angiotensinase C) . Similarity to both serine carboxypeptidase and prolylendopeptidase families; Tan F et al.; Prolylcarboxypeptidase, a lysosomal serine carboxypeptidase, cleaves COOH-terminal amino acids linked to proline, as in angiotensin II and III and {des-Arg9} bradykinin . About 25% of the enzyme protein was sequenced, and the complete sequence was deduced from its human kidney cDNA . The cDNA insert contained an open reading frame of 1488 base pairs coding for a protein of 496 residues . The authentic NH2-terminal sequence matched the deduced protein sequence starting with residue 46, suggesting the presence of both a signal and propeptide . The mature enzyme (451 residues) has a calculated M(r) = 51,043, whereas the M(r) of the purified glycoprotein is 58,000, indicating 12% carbohydrate . The overall sequence identity with serine peptidases is low (10-18%), but sequences around residues of the putative catalytic triad (Ser134, Asp333, His411) are similar (30-67%) to both the serine carboxypeptidases (e.g . deamidase or lysosomal protective protein, yeast carboxypeptidase Y, and KEX1 gene product) and the prolylendopeptidase family . Thus, prolylcarboxypeptidase links these two families, suggesting an evolutionary relationship . It is inhibited (Ki = 2.6 x 10(-7) M) by benzyloxycarbonyl-Pro-prolinal, a specific inhibitor of prolylendopeptidase, another angiotensin metabolizing enzyme . Prolylcarboxypeptidase contains serine or threonine residues repeated as the 26th residue 7 out of 9 times, with identical or similar amino acids in other positions in the repeats . The KEX1 gene product contains a similar motif, with serine or threonine as every 27th residue . The importance of prolylcarboxypeptidase is strongly suggested by its presence in various organs and cells and by the substrates it cleaves.

Biochemistry, 1993 Aug 3, 32(30), 7610 - 6
A highly sensitive probe for guanine N7 in folded structures of RNA: application to tRNA(Phe) and Tetrahymena group I intron; Chen X et al.; A nickel complex has been shown to promote conformation-specific oxidation of guanosine in polynucleotide RNA . In all cases, reaction was strictly dependent on the solvent exposure and surface properties of guanine N7 . Modification of native tRNA(Phe) (yeast) was detected at G18, G19, G20, and Gm34 and concurred with predictions based on its crystal structure . Additional guanine derivatives became exposed to oxidation only after the tRNA unfolded in the absence of Mg2+ . Reaction of the Tetrahymena group I intron RNA (L-21 ScaI) also compared favorably to its three-dimensional model by appropriately identifying guanosine residues in hairpin loops, duplex termini, and the essential cofactor binding site . These results complemented prior data generated by hydroxyl radical, and in combination they served to distinguish the solvent accessibility of sugar backbone and base positions in guanosine residues . Most importantly, this nickel complex exhibited greater selectivity than either dimethyl sulfate or RNase T1 for characterizing tRNA(Phe) and intron RNA.

Genomics, 1993 Aug, 17(2), 456 - 62
Isochores and CpG islands in YAC contigs in human Xq26.1-qter; Pilia G et al.; GC levels were assessed at 37 loci across 30 Mb of Xq26.1-qter, a region physically mapped in overlapping yeast artificial chromosome clones . In 8 Mb of R band Xq26, GC is relatively high (up to 44%) in the proximal 4 Mb and relatively low (40-41%) in the distal 4 Mb . Consistently low GC values (38-41%) are observed in G band Xq27 . In contrast, further toward the telomere in Xq28, the GC level rises progressively to reach 52% at 2 to 4 Mb from the end of the chromosome; this region is delimited by low GC loci . Across these regions of Xq, the content of rare-cutter restriction enzyme sites containing CpG, including "CpG islands" in the most completely mapped Xq26-27.1 region, is correlated with GC level . Isochore mapping can thus provide one index of putative gene content across mapped regions.

Genome, 1993 Aug, 36(4), 676 - 85
A developmental and molecular analysis of Cdc2 mutations in Drosophila melanogaster; Clegg NJ et al.; In fission yeast, the product of the cdc2 gene is required both for entry into S phase and mitosis . Homologs of cdc2 have been isolated from a number of metazoans, but in general they have not been amenable to genetic analysis . Here we describe P element transposon tagging of Cdc2 in Drosophila melanogaster and the analysis of 10 Cdc2 mutants . The recessive lethality of Cdc2P is associated with a P element located in the 5' untranslated region of the gene . Seven other alleles have unique single base pair substitutions in the coding region of Cdc2 . One allele, Cdc2B47, is mutated in the splice donor site of exon 1 . Most mutations in Cdc2, including the presumptive null allele Cdc2B47, die at the pupal stage, suggesting that the maternally supplied Cdc2 gene product drives earlier cell divisions . The phenotypes of our mutants are consistent with a role for Cdc2 in cell proliferation; however, we did not observe any perturbation of the endoreduplication cycle associated with the acquisition of polyteny.

Hum Mol Genet, 1993 Aug, 2(8), 1235 - 43
Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions . snU3 may be a candidate gene for the Smith-Magenis syndrome; Chevillard C et al.; The juxtacentromeric region of the human chromosome 17 short arm (17p11.2-p12) contains genes involved in the Charcot-Marie-Tooth type 1A disease (CMT1A) and the Smith-Magenis syndrome (SMS) . CMT1A is associated with a duplication of a short segment whereas SMS is linked to microdeletions, extending toward the centromere . We describe the construction and analysis of a 5 Mb YAC contig spanning the CMT1A duplicated segment and the distal part of four SMS microdeletions . We concluded that the YAC contig contains about 1Mb of genomic DNA which is deleted in the four SMS patients analysed . Moreover two YACs contain both STS deleted in SMS (U3) and STS duplicated in CMT1A (5H5), but the proximal breakpoint associated with the CMT1A duplication is not the same as the distal SMS breakpoint we studied . Finally we located five new STS in SMS deletion . Two of them, a microsatellite (D17S805(23)) and the gene coding for small nuclear RNA U3, have been localized in the contig we described . We may also note that snU3 is the first expressed sequence localized in an SMS deletion so far . The possible participation of this gene in the SMS phenotype is discussed.

Hum Mol Genet, 1993 Aug, 2(8), 1161 - 7
A contig of non-chimaeric YACs containing the spinal muscular atrophy gene in 5q13; Francis MJ et al.; We have constructed a contig of non-chimaeric yeast artificial chromosomes (YACs) across the candidate region for childhood autosomal recessive spinal muscular atrophy (SMA) in 5q13 . A novel microsatellite reduces the candidate region to approximately 400kb of DNA distal to D5S435 . The candidate region contains blocks of chromosome 5 specific repeats which have copies on 5p as well as elsewhere on 5q . Restriction mapping of the YACs reveals at least one CpG island in the SMA gene region . The YAC maps indicate that the contig contains minimal rearrangements or deletions . The data show the value of screening several YAC libraries simultaneously in order to construct a set of overlapping sequences suitable for candidate gene searches and direct genomic sequencing.

Hum Mol Genet, 1993 Aug, 2(8), 1105 - 15
2.6 Mb YAC contig of the human X inactivation center region in Xq13: physical linkage of the RPS4X, PHKA1, XIST and DXS128E genes; Lafreniere RG et al.; X chromosome inactivation is a mechanism of dosage compensation that regulates the expression of mammalian X-linked genes between XY males and XX females . This phenomenon is cis-acting, clonally heritable, and requires the presence of an X inactivation center (XIC) . In our attempts to characterize this phenomenon, we have focused on the physical organization of the human XIC localized to Xq13 . From previous studies, we had determined that the candidate XIC interval contained two loci (DXS128 and XIST) and was bound by the breakpoints of two structurally abnormal inactivated X chromosomes, a t(X;14) and an idic(Xp) . Here we present a refined mapping of the XIC-containing region using the breakpoint of a late replicating rearranged X (rea(X)), and the initial characterization of a set of 40 yeast artificial chromosomes (YACs) derived from the XIC-containing region . These YACs form a 2.6 Mb contig which completely covers the XIC, and physically links the RPS4X, PHKA1, XIST, and DXS128E genes, as well as a laminin receptor pseudogene (LAMRP4) . Furthermore, we have determined the relative orientations of these four genes, and have derived a restriction map of the region using the rare cutter enzymes BssHII, EagI, MluI, NruI, SalI, SfiI, SstII (or SacII), and NotI . We have identified at least 9 CpG-rich islands within this region, and have discovered a large (approximately 125 kb) inverted duplication proximal to the XIC based on symmetrical restriction patterns and homologous probes . We estimate the maximum size of the XIC-containing interval to be between 680 kb and 1200 kb, based on the localization of the breakpoints of the rearranged X chromosomes mentioned above . This lays the groundwork for the further characterization of the XIC region and the isolation of other expressed sequences therefrom.

Hum Mol Genet, 1993 Aug, 2(8), 1099 - 104
The interleukin-2 receptor gamma chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1; Puck JM et al.; The gene encoding the gamma chain of the lymphocyte interleukin-2 receptor has been cloned and shown to be required to associate with the beta chain in order for IL-2 internalization and cell activation to occur (1) . We considered this gene, IL2RG, a candidate for the X-linked form of severe combined immunodeficiency at the SCIDX1 locus, in which affected males have impaired lymphocyte development . Using fluorescence in situ hybridization and PCR amplification of somatic cell hybrid DNAs, we mapped IL2RG to human Xq13.1, a location within the SCIDX1 critical region established by linkage analysis . The 4.2 kb IL2RG gene was sequenced, and its genomic organization was elucidated . Seven of 19 transformed B-lymphocyte cell lines with independent SCIDX1 mutations had absent or minimal IL2RG mRNA . Unique point mutations were documented to be specifically associated with the disease and the carrier state in four unrelated affected males and their family members: one in a boy with no detectable IL2RG mRNA, in which the mutation ablated a splice donor site; one causing premature chain termination; and two causing distinct amino acid changes . The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCIDX1 gene is IL2RG . Noguchi et al . (2) have independently published IL2RG mapping to Xq13 and discovery of mutations in three affected males . The specific pathogenesis of IL2RG mutations and approaches to gene therapy can now be addressed in the X-linked form of SCID.

J Gen Virol, 1993 Aug, 74 ( Pt 8), 1633 - 8
African swine fever virus thymidylate kinase gene: sequence and transcriptional mapping; Yanez RJ et al.; A putative thymidylate kinase gene of African swine fever virus has been identified at the left end of the SalI I' fragment of the virus genome . The gene, designated A240L, has the potential to encode a protein of 240 amino acids with an M(r) of 27,754 and is transcribed early after infection . Primer extension analysis indicates that transcription is initiated a short distance from the first ATG codon of open reading frame A240L . The deduced amino acid sequence of this open reading frame shows significant similarity with the human, yeast and vaccinia virus thymidylate kinases, the degree of identity being 23.7, 25 and 23.5%, respectively . The putative African swine fever virus thymidylate kinase sequence is essentially collinear with the other thymidylate kinase sequences, but contains a carboxy-terminal extension of 37 amino acids rich in glutamic and aspartic acids . The A240L protein conserves the ATP-binding and nucleotide/nucleoside-binding domains characteristic of thymidylate kinases.

J Egypt Soc Parasitol, 1993 Aug, 23(2), 535 - 43
Rate of ingestion of Culex pipiens (L.) larvae (Diptera: Culicidae) stimulated by nutritive substances; Rashed SS; Relative rates of ingestion of fourth instar larvae of Culex pipiens as indicated by the number of substrate filled gut segments per unit time were determined and compared after exposure to nutritive and inert particles . Food particles with nutritive values (yeast; dried green algae, Chlamydomonas reinhordtii; wheat flour; fishmeal and corn flour) were ingested approximately 3-4 times faster than inert particles (koalin, talk, charcoal and chalk) . Aqueous extracts of yeast or dried algae accelerated ingestion of inert particles (in laboratory and in larval natural habitat) to the level of ingestion of nutritive particles, demonstrating gustatory stimulation of larvae . In the subsequent time span, ingestion rates of food particles decreased continuously with satiation of larvae.

Genetics, 1993 Aug, 134(4), 1119 - 34
The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism; Bazinet C et al.; The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles . As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches . Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction . Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe . The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs . Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome . A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation . This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group . Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage . A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood . Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline . In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny . However, hemizygous Chc4 males were invariably sterile . The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element . These findings suggest a specialized role for clathrin in spermatogenesis.

Plant Mol Biol, 1993 Aug, 22(5), 931 - 5
Comparative sequence analysis of cis elements present in Glycine max L . leghemoglobin lba and lbc3 genes; She Q et al.; The soybean leghemoglobin lba gene promoter sequence was determined and aligned with the promoter sequence of the soybean lbc3 gene from the same gene family . Five highly conserved regions were found . There are two large conserved regions, one of which overlaps the basic promoter while the other defines a minimal enhancer in the upstream positive elements . Within the minimal enhancer, an inverted repeat with similarity to the binding site of a yeast transcription factor, GCN4, was found . This particular repeat is conserved in the promoters of all functional soybean lb genes as well as in lb gene promoters from other legumes . This suggests that the inverted repeat is important for leghemoglobin gene expression.

Mol Gen Genet, 1993 Aug, 240(2), 265 - 72
Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone; Kleine M et al.; We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L . cv . Franka) using the vector pYAC4 . The library consists of approximately 18,000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome . Size fractionation after ligation resulted in an increased average insert size (av . 370 kb) but also in a substantial decrease in cloning efficiency . Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe . Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique . A single YAC, designated Y66C11, with a 120 kb insert was isolated . This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe . Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 6914 - 8
E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product; Flemington EK et al.; Previous studies have shown that the carboxyl-terminal region of E2F-1 (residues 368-437) can support transcriptional activation when linked to the DNA-binding domain of the yeast transcription factor GAL4 . This region also contains an 18-residue retinoblastoma (RB)-binding sequence, raising the possibility that RB binding might inhibit the ability of E2F-1 to form protein-protein contacts required for activation . Here we report a further analysis of the E2F-1 activation domain . In addition, we show that overexpression of RB, but not the RB mutant, RBd22, can inhibit GAL4/E2F-1 activity in vivo . Moreover, expression of the simian virus 40 large tumor antigen (T antigen), but not the RB-binding defective T antigen point mutant, K1, can overcome this repression . Three different GAL4/E2F-1 mutants that activate transcription, but fail to bind to RB, are not significantly affected by overexpression of RB . These findings support a model wherein RB suppresses E2F-1-mediated transcriptional activation through direct physical association.

Mol Cell Biol, 1993 Aug, 13(8), 4485 - 93
3' splice site selection in dicot plant nuclei is position dependent; Lou H et al.; In contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mRNAs is poorly understood . Splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes . Perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons . To define elements critical for 3' splice site selection in dicotyledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by using an autonomously replicating plant expression vector . Using a series of intron rearrangements which reposition the 3' intron-exon border, we demonstrate that the normal 3' splice site is defined in a position-dependent manner and that cryptic 3' splice sites within the intron are masked by the presence of a functional downstream 3' splice site . Disruption of the AU-rich elements upstream from the normal 3' splice site indicates that multiple AU elements between -66 and -6 cooperatively define the 3' boundary of the intron . These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU transition points . Functional 3' splice sites located downstream from these AU-rich sequences are preferentially selected over sites embedded within them.

Curr Opin Cell Biol, 1993 Aug, 5(4), 653 - 60
Microfilaments and membranes; Bretscher A; Microfilaments are intimately involved in many plasma and internal membrane functions . Recent studies of microfilament-membrane linking proteins and non-filamentous myosins implicate microfilaments in diverse functions, including transmembrane signaling and vesicular transport . Evidence from animal and yeast cells suggests that microfilaments are regulated by protein phosphosphorylation, small GTP-binding proteins and associations involving SH3 domains.

Mol Biochem Parasitol, 1993 Aug, 60(2), 161 - 70
Molecular cloning of a rac family protein kinase and identification of a serine/threonine protein kinase gene family of Entamoeba histolytica; Que X et al.; Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases . These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells . One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene (Eh rac1) from an E . histolytica genomic library . The open reading frame of Eh rac1 was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain . The catalytic domain of Eh rac1 was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs . Southern blots of ameba DNA showed that the Eh rac1 gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh rac1 mRNAs than did nonpathogenic amebae . These studies suggest that E . histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.

Mol Endocrinol, 1993 Aug, 7(8), 1041 - 8
Glucose regulation of transforming growth factor-alpha expression is mediated by products of the hexosamine biosynthesis pathway; Daniels MC et al.; We have recently shown that glucose and glucosamine regulate the transcription of transforming growth factor-alpha (TGF alpha) in rat aortic smooth muscle (RASM) cells . Based on the increased potency of glucosamine compared to glucose, we hypothesized that stimulation of TGF alpha transcription by glucose is mediated through the hexosamine biosynthesis pathway . The yeast cDNA for the rate-limiting enzyme of this pathway, glutamine:fructose-6-phosphate amidotransferase (GFA), was therefore expressed in RASM cells . GFA-transfected cells showed an increase in GFA activity, exhibiting a 2.2-fold increase in the synthesis of glucosamine-6-phosphate, the first product of the hexosamine biosynthetic pathway . To test the effect of GFA overexpression on TGF alpha transcriptional activity, cells were transiently cotransfected with GFA along with a reporter plasmid containing the firefly luciferase gene under control of the TGF alpha promoter . GFA-transfected cells exhibited a glucose-dependent 2-fold increase in TGF alpha activity compared to control cells . Maximal stimulation of TGF alpha-luciferase activity by glucosamine, however, was equivalent in GFA-and control-transfected cells, confirming that the stimulation observed by both agents operated through the same pathway . This increase in TGF alpha activity was inhibited (85% at 0.5 mM glucose and 69% at 30 mM glucose) by the glutamine analog and inhibitor of GFA, 6-diazo-5-oxonorleucine (10 microM) . Control studies confirmed that the increased TGF alpha-luciferase activity in the GFA-expressing cells was not an artifact of altered growth, survival, or transfection efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Sci, 1993 Aug, 105 ( Pt 4), 1101 - 13
Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei . Divergent ER retention signals in a lower eukaryote; Bangs JD et al.; Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified . A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein . gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers . Trypanosomes are the most primitive eukaryote yet in which BiP has been identified . The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy . The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum . The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al . (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

Plant J, 1993 Aug, 4(2), 279 - 93
A plant gene with homology to D-myo-inositol-3-phosphate synthase is rapidly and spatially up-regulated during an abscisic-acid-induced morphogenic response in Spirodela polyrrhiza; Smart CC et al.; The induction of turions (dormant vegetative buds) by the plant growth regulator abscisic acid (ABA) in the aquatic angiosperm Spirodela polyrrhiza is one of the few examples of a plant hormone triggering a morphogenic response . In order to aid our understanding of the molecular basis of this phenomenon a cDNA library has been prepared from ABA-induced S . polyrrhiza and by differential screening a number of early ABA-up-regulated genes have been isolated . This paper reports the characterization of one such cDNA (tur1) from S . polyrrhiza which codes for a protein highly homologous to yeast D-myo-inositol-3-phosphate synthase (EC.5.5.1.4), the catalyst for the first committed step in inositol biosynthesis . This is the first reported cloning of a plant gene involved in inositol production . It is shown by Northern analysis and in situ hybridization that ABA induces a rapid preferential up-regulation of this gene and that this is localized to the stolon tissue . The stolon connects the developing turion to the node of the mother frond (the meristematic region involved in the production of turions which arise as a morphogenic response to this hormone) . This increase in tur1 transcript level is accompanied by a rise in the activity of D-myo-inositol-3-phosphate synthase . The possible role of inositol in ABA-induced events during turion formation is discussed.

Anal Biochem, 1993 Aug 1, 212(2), 325 - 34
Arsenical-based affinity chromatography of vicinal dithiol-containing proteins: purification of L1210 leukemia cytoplasmic proteins and the recombinant rat c-erb A beta 1 T3 receptor; Kalef E et al.; Proteins containing vicinal dithiols were purified by affinity chromatography using Sepharose 4B linked to aminohexanoyl-4-aminophenylarsineoxide (As-Sepharose) . The protein vicinal dithiols form stable dithioarsine derivatives with the arsine oxide moieties of the gel . The adsorbed proteins were eluted, at physiological pH, by buffers containing beta-mercaptoethanol or dithiothreitol . The dithiol proteins were identified by their specific labeling with N-iodoacetyl-3-{125I}-iodotyrosine . Cytoplasmic thiol proteins of L1210 murine leukemia lymphoblasts were separated into three classes by interaction with As-Sepharose . Proteins that did not bind to the gel consisted of monothiol proteins; proteins eluted by beta-mercaptoethanol include vicinal dithiol-containing proteins with low affinity for the arsine oxide . DL-Dithiothreitol (DTT) elutes a large group of vicinal dithiol-containing proteins with high affinity for the arsine groups . Gradient elution allowed characterization of the relative affinities of dithiol proteins for the As-Sepharose . A one-step purification of the L-triiodothyronine recombinant rat c-erb A beta 1 T3 receptor synthesized in yeast required pretreatment with DTT for binding to As-Sepharose and resulted in a 62-fold increase in specific activity . The procedure allows purification of proteins inhibited by phenylarsine oxide such as phosphotyrosine phosphatases, proteins that are subject to redox regulation, and dithiol proteins that are targets of oxidative stress.

Toxicol Lett, 1993 Aug, 69(2), 121 - 7
In vitro inactivation of glucose-6-phosphate dehydrogenase from human red blood cells by acrolein: a possible biomarker of exposure; Trieff NM et al.; We have investigated the possibility of utilizing glucose-6-phosphate dehydrogenase (G6PD) as a macromolecular (biological) marker of acrolein exposure . The result showed a dose-dependent inactivation of the erythrocyte G6PD in situ or as a purified enzyme from human erythrocytes or yeast . Amino acid analysis on the chemically modified yeast G6PD showed a formation of a lysine adduct which is probably linked to the inactivation.

J Hosp Infect, 1993 Aug, 24(4), 245 - 59
Selective decontamination with nystatin for control of a Candida outbreak in a neonatal intensive care unit; Damjanovic V et al.; Selective decontamination of the digestive tract (SDD) with oral nystatin was evaluated as a measure to control an outbreak of Candida infection in a neonatal intensive care unit (NICU) . Seventy-six out of 106 neonates who carried Candida spp . received the main study manoeuvre (the application of oral nystatin in the throat and stomach) during the 12-month open trial . One third of the neonates weighed < 1500 g whilst about half were being ventilated . The mean stay was 33.2 d (SD +/- 46.9) . Two cases with candidaemia within a fortnight were associated with a yeast carriage rate in the NICU of about 50%; more than 80% of the isolates were Candida parapsilosis . During the implementation period there were four new neonates with fungaemia caused by C . parapsilosis . Once the carriage rate dropped below 5% (P < 0.001), no new cases of systemic infection with the outbreak strain were recognized in the following 8 months . It took 3.5 months to control the outbreak . The observation that all other clinical diagnostic samples were free from Candida suggests that translocation from throat or gut into the systemic circulation occurred . SDD with oral nystatin was effective in reducing the yeast carriage index (mean index 1.93, before SDD; 0.45, after SDD; P < 0.001) . A significant reduction of carriage, both in rates and indices, is thought to have contributed to the control of this candida outbreak.

Genome, 1993 Aug, 36(4), 712 - 24
The genetic and RFLP characterization of the left end of linkage group III in Caenorhabditis elegans; Pilgrim D; A genetic approach was taken to identify new transposable element Tc1-dependent polymorphisms on the left end of linkage group III in the nematode Caenorhabditis elegans . The cloning of the genomic DNA surrounding the Tc1 allowed the selection of overlapping clones (from the collection being used to assemble the physical map of the C . elegans genome) . A contig of approximately 600-800 kbp in the region has been identified, the genetic map of the region has been refined, and 10 new RFLPs as well as at least four previously characterized genetic loci have been positioned onto the physical map, to the resolution of a few cosmids . This analysis demonstrated the ability to combine physical and genetic mapping for the rapid analysis of large genomic regions (0.5-1 Mbp) in genetically amenable eukaryotes.

J Clin Microbiol, 1993 Aug, 31(8), 2124 - 33
Oligonucleotide fingerprinting of isolates of Candida species other than C . albicans and of atypical Candida species from human immunodeficiency virus-positive and AIDS patients; Sullivan D et al.; Oligonucleotide fingerprinting of genomic DNA from oral isolates of four different Candida species other than C . albicans and atypical chlamydospore-positive isolates from human immunodeficiency virus (HIV)-positive individuals and AIDS patients was investigated as a means for differentiating between isolates within individual species . Oligonucleotides composed of simple repetitive sequence motifs, including (GACA)4, (GATA)4, (GGAT)4, (GTG)5, and (GT)8, all yielded fingerprints suitable for strain segregation of 8 C . tropicalis isolates, 12 Torulopsis (Candida) glabrata isolates, 8 atypical Candida isolates, and, except for (GATA)4, 2 C . krusei probe in turn and so generate several distinct DNA fingerprints of the same DNA sample . However, none of the probes yielded fingerprints suitable for strain segregation with three C . parapsilosis isolates . The (GATA)4 probe was also used to detect restriction fragment length polymorphisms among a genetically closely related group of atypical Candida isolates on primary isolation from an additional HIV-infected patient . These chlamydospore-positive atypical Candida isolates were sucrose positive, were of C . albicans serotype A, hybridized weakly with the C . albicans-specific mid-repeat sequence probe 27A, and yielded fingerprint profiles by random polymorphic DNA analysis that were distinct from those derived from C . albicans isolates . The C . stellatoidea ex-type strain NCPF 3108 was indistinguishable from the atypical Candida isolates in all these tests and also yielded an identical carbohydrate and nitrogen source assimilation profile by using the ID 32C yeast identification system.

Hum Genet, 1993 Aug, 92(1), 95 - 9
Physical mapping shows close linkage between the alpha-galactosidase A gene (GLA) and the DXS178 locus; Vetrie D et al.; X-linked agammaglobulinaemia (XLA) is an inherited disorder characterised by a lack of circulating B-cells and antibodies . While the gene involved in XLA has not yet been identified, the locus for the disorder is tightly linked to the polymorphic marker DXS178, which maps to Xq22 . Fabry disease is an X-linked recessive disorder caused by a deficiency in the lysosomal enzyme alpha-galactosidase A . The gene encoding this enzyme has been characterized and also maps to Xq22 . Using pulsed field gel electrophoresis we have constructed a long-range restriction map that shows that the alpha-galactosidase A gene (GLA) and DXS178 lie no more than 140 kb apart on a stretch of DNA containing a number of putative CpG islands . We have also isolated yeast artificial chromosome (YAC) clones that confirm this physical linkage . The localisation of DXS178 near the alpha-galactosidase A gene will facilitate carrier detection in Fabry families using restriction fragment length polymorphism (RFLP) analysis . The identification of a number of CpG islands near DXS178 also provides candidate locations for the gene responsible for XLA.

Curr Opin Biotechnol, 1993 Aug, 4(4), 484 - 9
Peroxidases; Poulos TL; The availability of recombinant cytochrome c peroxidase from yeast has already proved to be of considerable importance in developing the protein engineering of peroxidases and of metalloproteins in general . With the recent increase in information on peroxidase sequences, further developments in recombinant expression systems and the determination of an increasing number of peroxidase crystal structures, it should soon be possible to make significant advances in engineering peroxidases for biotechnological purposes.

Inflammation, 1993 Aug, 17(4), 499 - 509
Terpenes enhance metabolic activity and alter expression of adhesion molecules (Mac-1 and L-selectin) on human granulocytes; Johard U et al.; Granulocytes from healthy blood donors were exposed to terpenes dissolved in ethanol . Flow cytometry was used to measure the expression of the cell surface receptors Mac-1 L-selectin, and CR1, which were detected by monoclonal antibodies . The phagocytic activity was determined by using C3b-coated yeast particles as prey . The metabolic activity was measured by determining the intracellular hydrogen peroxide production using dichlorofluorescein diacetate . After terpene exposure the expression of Mac-1 and CR1 increased (P < 0.001 and P < 0.01, respectively) and the expression of L-selectin decreased (P < 0.001) . There was also an increased metabolic activity (P < 0.001) . The cell viability, the cell count, and the phagocytic activity remained unchanged . These findings suggest that terpene exposure triggers the granulocytes to a higher degree of activation in terms of an altered expression of the adhesion molecules and an increased metabolic activity.

Genomics, 1993 Aug, 17(2), 393 - 402
Generation of a human chromosome 18-specific YAC clone collection and mapping of 55 unique YACs by FISH and fingerprinting; Chang E et al.; A yeast artificial chromosome (YAC) library was constructed from a somatic cell hybrid line in which the human chromosome content had been reduced by repeated subcloning to one or two copies of chromosome 18 . Screening of 4700 primary yeast transformants generated 74 clones containing a YAC with a human DNA insert averaging 190 kb in size . The human YACs were localized to subregions of chromosome 18 by in situ hybridization of biotin-labeled Alu-PCR products obtained using total yeast DNA as a template . Comparisons of interspersed repetitive sequence-PCR and restriction fragment fingerprint patterns identified five sets of identical and three sets of overlapping YACs . Dual-label fluorescence in situ hybridization interphase mapping was used to determine the order of some nonoverlapping YACs . STS (sequence-tagged site) content mapping was carried out with PCR primers for 56 chromosome 18-specific markers . The identification of YACs containing four known genes--encoding the pituitary adenylate cyclase activating polypeptide (PA-CAP), the myelin basic protein (MBP), ferrochelatase (FECH), and SSAV1, an endogenous retroviral element related to the SSAV virus--provides a precise cytological map position for the respective loci . Our final collection of 55 randomly isolated, unique, and regionally localized YACs (D18S107-D18S161) is distributed over the entire chromosome and collectively covers approximately 12 Mb, i.e., 16% of the estimated 77 Mb of DNA in euchromatin of chromosome 18 . These YACs provide reagents for the isolation of genes in these regions and represent nucleation points for the generation of STS to increase coverage of the chromosome by YAC contigs.

Biochemistry, 1993 Jul 27, 32(29), 7388 - 95
Secondary structure of a pair of fibronectin type 1 modules by two-dimensional nuclear magnetic resonance; Williams MJ et al.; The fourth and fifth type 1 module pair, corresponding to residues 151-244 from the amino terminus of human fibronectin, has been produced as a recombinant protein using a yeast expression system and studied by two-dimensional homonuclear 1H nuclear magnetic resonance (NMR) spectroscopy . The sequence-specific resonance assignment of the 1H NMR spectrum has been completed using a combination of 2D 1H nuclear Overhauser effect (NOE) spectroscopy, homonuclear Hartmann-Hahn, and correlated spectroscopy spectra recorded under a variety of pH and temperature conditions . Slow exchanging amide protons have been identified and estimates of many backbone 3JNH-C alpha H coupling constants were obtained by line shape fitting . The secondary structures of each module conform closely to the "consensus" fibronectin type 1 module structure determined previously for two other single type 1 modules . In the module pair described here, the two modules are linked by a short five-residue linker which appears to form a turn . The intermodule interface is defined by NOEs observed between a hydrophobic three-residue sequence from the fourth type 1 module and residues in the first double-stranded beta-sheet of the fifth type 1 module . The interaction is dominated by a tryptophan residue (unconserved in other type 1 sequences) within the fourth module, which causes large upfield ring current shifts for several proton resonances from the beta-sheet of the fifth module . The NMR data indicate that there is little or no relative reorientation of the two modules about the linker region but rather that the two modules combine with a fixed and intimate hydrophobic contact.

J Biol Chem, 1993 Jul 25, 268(21), 16074 - 81
Human salivary endo-beta-N-acetylglucosaminidase HS specific for complex type sugar chains of glycoproteins; Ito K et al.; The enzyme that catalyzed the conversion of human salivary alpha-amylase family A (HSA-A) to family B (HSA-B) was identified . It was partially purified from the precipitate obtained by centrifugation of human saliva at 105,000 x g for 60 min by solubilization with 3{(3-cholamidopropyl)dimethylammonio}-1-propane sulfonate and column chromatographies with Sephacryl S-300-HR and hydroxylapatite . The enzyme preparation was practically free from contaminating exoglycosidases and proteases . The enzyme cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of HSA-A, as shown by the isolation of the protein moiety which contained 1 GlcNAc and 1 Fuc residue and the sugar chain (Gal)2(Fuc)1(GlcNAc)2(Man)3(GlcNAc) . This enzyme also cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of human transferrin tetraglycopeptide Asn-Tyr-Asn(GlcNAc)2(Man)3(GlcNAc)2(Gal)2-Lys to yield equimolar amounts of peptide Asn-Tyr-Asn(GlcNAc)Lys and sugar chain (Gal)2(GlcNAc)2(Man)3(GlcNAc) . The enzyme was identified as an endo-beta-N-acetylglucosaminidase . The enzyme acted on HSA-A with desialylated and defucosylated outer chain moieties of the sugar chains at a similar rate as that of native HSA-A . The enzyme activity was reduced to 13 and 5% using HSA-A with the sugar chains whose outer chain moieties lacked Gal and GlcNAc, respectively, from the nonreducing end . The enzyme also acted on human transferrin, calf fetuin, and asparagine oligosaccharides of transferrin and fetuin . On the other hand, the enzyme did not act on ovalbumin, RNase B, Taka-amylase, yeast invertase, and ovalbumin asparagine oligosaccharides . These results indicate that human salivary endo-beta-N-acetylglucosaminidase is specific for complex type sugar chains and can release the sugar chains from native glycoproteins and glycopeptides regardless of the existence of a Fuc residue on the proximal GlcNAc of the N,N'-diacetylchitobiose core of their sugar chains . The source of the enzyme was epithelial cells peeling from the oral cavity epithelium into saliva . The enzyme was thought to be integrated on the surface of the epithelial cell membrane . This enzyme was named endo-beta-N-acetylglucosaminidase HS . Thus, these studies indicate that the properties of the enzyme are distinct from those of known endo-beta-N-acetylglucosaminidase and endo-beta-N-acetylglucosaminidase HS is a novel endo-beta-N-acetylglucosaminidase.

Biochem J, 1993 Jul 15, 293 ( Pt 2), 595 - 9
Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase . Correlation between equilibrium dialysis binding and enzyme kinetic data; Molnar M et al.; The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis . Whereas the Kd of ATP binding varies between 0.17 and 0.23 mM (S.E.M . 0.03 mM) for both enzymes, independently of the presence of Mg2+, the Kd values for ADP and MgADP binding are in the range 0.18-0.27 mM (S.E.M . 0.04 mM) and 0.05-0.06 mM (S.E.M . 0.01 mM) respectively . Thus Mg2+ exclusively tightens the interaction of ADP, but not of ATP, with the protein molecule . Although the equilibrium dialysis data are consistent with a model possessing a single site for nucleotides, the existence of a much weaker secondary site (with a Kd value at least two orders of magnitude larger) cannot be excluded . The binding of AMP and adenosine to pig muscle 3-phosphoglycerate kinase is weaker than binding of MgATP; the respective Kd values are 0.36 +/- 0.05 mM and 0.65 +/- 0.05 mM . Thus, in addition to the interaction of the alpha-phosphate that is detectable by crystallography {Banks, Blake, Evans, Haser, Rice, Hardy, Merrett and Phillips (1979) Nature (London) 279, 773-777}, the beta- and/or gamma-phosphate(s) of MgATP may also interact with the enzyme molecule . The fact that MgADP binds more tightly than ADP is consistent with its stronger inhibition of the reaction catalysed by the enzyme between 3-phosphoglycerate and MgATP . MgADP is a product of this reaction, and inhibits it competitively with both substrates; as an inhibitor its KI is comparable with the Kd found in binding studies . At the same time, the Km value for MgADP in the reverse reaction (0.18 +/- 0.05 mM; mean +/- S.E.M.) is higher than these constants; this may be due either to a different kinetic mechanism in this direction of the enzymic reaction, or to different binding modes of MgADP as inhibitor and as substrate . The reason why inhibition by MgADP is competitive with 3-phosphoglycerate may be that its binding prevents the specific change in conformation that the enzyme undergoes {Harlos, Vas and Blake (1992) Proteins 12, 133-144} when it binds 3-phosphoglycerate.

Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6686 - 90
Differential organ-specific expression of three poly(A)-binding-protein genes from Arabidopsis thaliana; Belostotsky DA et al.; Poly(A)-binding protein (PABP) is considered an essential component of a eukaryotic cell; deletion of the PABP-coding gene in yeast leads to a lethal phenotype . PABP is implicated in numerous aspects of posttranscriptional regulation, including mRNA turnover and translational initiation . A nested set of degenerate PCR primers designed from regions conserved among yeast, Xenopus, and human PABP sequences was used to amplify genomic DNA fragments from Arabidopsis thaliana . Hybridization screening of genomic and cDNA libraries with a genomic PCR probe led to the isolation of three diverse Arabidopsis genes encoding PABPs, PAB1, PAB3, and PAB5 . All three sequences contain the expected four RNA-recognition motifs . Sequence diversity between these genes equals or exceeds the diversity among animal and fungal sequences . One of the genes, PAB5, and its cDNA were completely sequenced . Its open reading frame encodes a 73.2-kDa protein containing a number of amino acid motifs characteristic of PABPs from different species . Moreover, in vitro synthesized PAB5 protein bound to poly(A)-Sepharose with high specificity . All three genes isolated showed organ-specific patterns of expression . PAB5 and PAB3 RNAs were detected only in floral organs, with the highest level of expression in immature flowers . PAB1 RNA was observed predominantly in roots, was less abundant in immature flowers, and was not detected in any other organ examined (stems, leaves, mature flowers, siliques) . This suggests a potentially unique role for PABPs in organ-specific posttranscriptional regulation in plants.

J Biol Chem, 1993 Jul 15, 268(20), 15056 - 60
Tau protein kinase II has a similar characteristic to cdc2 kinase for phosphorylating neurofilament proteins; Hisanaga S et al.; Tau protein kinase II purified from a bovine brain tau protein fraction (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T., and Imahori, K . (1992) J . Biol . Chem . 267, 10897-10901) was shown to have a similar substrate specificity to cdc2 kinase in that both phosphorylate neurofilament (NF) proteins . Tau protein kinase II recognized the dephosphorylated form of the heavy subunit of NF (NF-H) as a predominant substrate . The substrate was phosphorylated to the same extent with tau protein kinase II as with cdc2 kinase . Upon phosphorylation, the electrophoretic mobility of the NF-H on SDS-polyacrylamide gel electrophoresis changed to the position of the phosphorylated form . A synthetic peptide containing a KSPXK sequence was by far a better substrate for tau protein kinase II than that containing a KSPXX sequence, as was also observed with cdc2 kinase . NF-H lost its microtubule-associating ability upon phosphorylation with tau protein kinase II as well as with cdc2 kinase . Although anti-PSTAIR antibody (PSTAIR is an amino acid sequence commonly found in cdc2 and several cdc2-related kinases) failed to react with tau protein kinase II, tau protein kinase II bound to p13suc1-Sepharose beads (p13suc1 is a yeast protein known to bind to cdc2 kinase).

J Biol Chem, 1993 Jul 15, 268(20), 14724 - 31
High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors; Basse CW et al.; We have previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato cells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the high mannose oligosaccharides released from the pure glycopeptide elicitors by endo-beta-N-acetylglucosaminidase H acted as suppressors of elicitor activity (Basse, C . W., Bock, K., and Boller, T . (1992) J . Biol . Chem . 267, 10258-10265) . One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxycarbonyl-L-{35S}methionine and purified by reversed phase high performance liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol . This radiolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as to microsomal membranes derived from these cells . Ligand saturation experiments, performed with microsomal membranes, gave a dissociation constant (Kd) of 3.3 nM as determined by Scatchard analysis . Various glycopeptide elicitors and preparations from yeast invertase were compared with respect to their abilities to compete for binding of 35S-labeled gp 8c to tomato membranes and to induce ethylene biosynthesis in tomato cells . These studies revealed a high degree of correlation between elicitor activities in vivo and displacement activities in vitro . In both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues . The high mannose oligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also . The suppressor-active glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very similar displacement activities, and the inhibitory constant (Ki) of the glycan Man11GlcNAc was very similar to the Kd value calculated for 35S-labeled gp 8c, indicating that the glycopeptide elicitors and the glycan suppressors derived from these elicitors competed with similar affinities for the same binding site . The suppressor-inactive glycan Man8GlcNAc had a 200-fold lower capacity to compete for binding of 35S-labeled gp 8c to tomato membranes compared with the suppressor-active glycan Man11GlcNAc . Our results demonstrate the existence of a specific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for induction of the stress response, respectively, by competing for this binding site.

Nucleic Acids Res, 1993 Jul 11, 21(14), 3217 - 26
Interlocked circle formation by group I introns: structural requirements and mechanism; Winter AJ et al.; Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro . Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal . A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron . Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron . Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence . The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction . Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation . The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions . It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot . We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles . The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.

Biochem Pharmacol, 1993 Jul 6, 46(1), 117 - 23
Metabolic activation of n-butyraldoxime by rat liver microsomal cytochrome P450 . A requirement for the inhibition of aldehyde dehydrogenase; DeMaster EG et al.; n-Butyraldoxime (n-BO) is known to cause a disulfiram/ethanol-like reaction in humans, a manifestation of the inhibition of hepatic aldehyde dehydrogenase (AIDH) . As with a number of other in vivo inhibitors of AIDH, n-BO does not inhibit purified AIDH in vitro, suggesting that a metabolite of n-BO is the actual inhibitor of this enzyme . In re-examination of the effect of n-BO on blood acetaldehyde levels following ethanol in the Sprague-Dawley rat, we found that pretreatment with substrates and/or inhibitors of cytochrome P450 blocked the n-BO-induced rise in blood acetaldehyde in the following order of decreasing potency: 1-benzylimidazole (0.1 mmol/kg) > 3-amino-1,2,4-triazole (1.0 g/kg) > ethanol (3.0 g/kg) > phenobarbital (0.1% in the drinking water, 7 days) > SKF-525A (40 mg/kg) . Rat liver microsomes were shown to catalyze the conversion of n-BO to an active metabolite that inhibited yeast AIDH . This reaction was dependent on NADPH and molecular oxygen and was inhibited by CO and 1-benzylimidazole . Hydroxylamine, postulated by others to be a metabolite of n-BO, inhibited AIDH via a catalase-mediated reaction and not through an NADPH-supported microsome-catalyzed reaction . Using GLC-mass spectrometry, 1-nitrobutane (an N-oxidation product) and butyronitrile (a dehydration product) were identified as metabolites from microsomal incubations of n-BO . However, neither of these metabolic products inhibited AIDH directly or in the presence of liver microsomes and NADPH . We conclude that another NADPH-dependent, cytochrome P450-catalyzed metabolic product of n-BO is responsible for the inhibition of AIDH by n-BO.

J Mol Biol, 1993 Jul 5, 232(1), 268 - 84
Solution structure of a pair of complement modules by nuclear magnetic resonance; Barlow PN et al.; A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR . The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology . In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H . Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface . Comparatively few nuclear Overhauser effects were observable between protons on different modules . Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e . rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data . However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt" . These observations may suggest upper limits on the relative flexibility of the two modules . Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Oncogene, 1993 Jul, 8(7), 1713 - 20
A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein; Madden SL et al.; The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-containing transcription factor (WT1) . WT1 binds to the consensus sequence (5'-GCGGGGGCG-3') and represses transcription when bound to this site in vivo . The mechanism of repression is not yet defined . To investigate the mechanisms of transcriptional repression and map the domains of WT1 responsible, we constructed hybrid proteins between the yeast GAL4 1-147 DNA binding domain and WT1 . Fusion of a 298 amino acid glutamine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding domain created a potent transcriptional repressor . The use of N- and C-terminal truncations of this segment demonstrated that as few as 96 amino acids were required for active repression by GAL4-WT1 hybrid proteins in NIH3T3 fibroblasts . However, the truncated GAL4-WT1 fusion proteins functioned poorly as repressors in embryonic kidney-derived 293 cells, suggesting cell type-specific requirements for transcriptional repression . Site-directed mutagenesis of the WT1 repression domain revealed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression function . Single repressor binding sites placed upstream of the transcription start site conferred WT1-mediated repression to a heterologous promoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression . Significant repression of transcription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transcription initiation . We conclude that the transcriptional repression function of WT1 is contained in the N-terminal, non-DNA binding domain of the protein and that repression can be functionally transferred to a heterologous DNA binding domain.

Genomics, 1993 Jul, 17(1), 98 - 105
A fluorescence in situ hybridization map of human chromosome 21 consisting of 30 genetic and physical markers on the chromosome: localization of 137 additional YAC and cosmid clones with respect to this map; Gingrich JC et al.; A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome . Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map . For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter . The location of the markers was established with a standard error of +/- 1.9 Mb using from 9 to 63 FL measurements for each probe . The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome . Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome . The cosmids, chosen at random from a flow-sorter chromosome 21 cosmid library, show some biases in chromosome distribution.

Genomics, 1993 Jul, 17(1), 52 - 8
YAC clone contigs surrounding the Zfx and Pola loci on the mouse X chromosome; Hamvas RM et al.; Pulsed-field mapping of a number of DNA markers in the Pola-Zfx region of the mouse X chromosome has established a genomic restriction map extending over 1.4 Mb . A number of YAC clones from the Pola-Zfx region have been isolated from three mouse YAC libraries--first, a mouse C57BL/10 partial R1 YAC library constructed in a yeast strain carrying a rad52 mutation (Chartier et al . (1992) Nature Genetics 1: 132-136); second, a mouse C3H partial R1 library (Larin et al . (1991) Proc . Natl . Acad . Sci . USA 88: 4123-4127); and third, a mouse C57BL/6 partial R1 library (Burke et al . (1991) Mamm . Genome 1:65) . Six YAC clones encompass the Zfx-Pola region, confirming the linkage of the Pola and Zfx loci and establishing a physical map order in this region of cen-Pola-DXCrc140-DXCrc57-Zfx-tel . The close linkage of Pola and Zfx in the mouse genome suggests that the POLA and ZFX loci must also be closely linked on the human X chromosome.

Genomics, 1993 Jul, 17(1), 25 - 32
Physical analysis of the terminal 240 kb of DNA from human chromosome 7q; Riethman HC et al.; DNA from a 240-kb human telomeric yeast artificial chromosome (HTY) clone was analyzed using physical mapping methods . Cosmid subclones of the YAC were fingerprinted using restriction enzyme digestion and human repeat sequence hybridization and then assembled into two contigs that together span 93% of the human insert . Data from restriction mapping and Bal31 exonuclease experiments indicate that, except for the truncation of distal genomic (T2AG3)n sequences, the molecular clone HTY 146 contains a contiguous, 230-kb telomere-terminal fragment from 7qter . Markers derived from this clone will allow telomeric closure of the physical and genetic linkage maps of human chromosome 7q.

Genomics, 1993 Jul, 17(1), 211 - 4
Fifty sequenced-tagged sites on human chromosome 11; Miwa T et al.; Fifty novel sequenced-tagged sites (STSs) were identified from cosmid clones mapped to human chromosome 11 . DNA sequences were determined for one or both cloning ends of 69 cosmid markers that had each been localized to 1 of 24 subchromosomal regions by means of hybridization to somatic cell hybrid panels . Proper primer sequences and appropriate conditions for a polymerase chain reaction (PCR) were determined for each marker . Twenty-one of the cosmids were not suitable for generating STSs, mainly because both of their ends contained repetitive elements such as Alu and L1 sequences; however, some were inappropriate because the sizes of their PCR products from human DNA, used as template, were same as those from yeast DNA . Finally, 50 STSs were established from 48 clones: 20 were derived from markers localized on the short arm and 30 from the long arm . These STSs can serve as new reagents for investigating human DNA in somatic cell hybrids and for isolating yeast artificial chromosomes to anchor large DNA contigs and fine-scale physical maps of chromosome 11.

Mol Biol Cell, 1993 Jul, 4(7), 747 - 56
A rab protein regulates the localization of secretory granules in AtT-20 cells; Ngsee JK et al.; Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles . Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment . In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles . When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles . Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes . These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH . Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation . The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.

Proc Natl Acad Sci U S A, 1993 Jul 1, 90(13), 5889 - 92
Signal transduction via the MAP kinases: proceed at your own RSK; Blenis J; An explosion of new information linking activation of cell surface signal initiators to changes in gene expression has recently emerged . The focus of much of this information has centered around the agonist-dependent activation of the mitogen-activated protein (MAP) kinases . Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals . Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.

J Virol, 1993 Jul, 67(7), 3868 - 76
The carboxy-terminal transcription enhancement region of the human spumaretrovirus transactivator contains discrete determinants of the activator function; Venkatesh LK et al.; The bel1 gene of human spumaretrovirus (HSRV) encodes a 300-amino-acid nuclear protein termed Bel1 that is a potent activator of transcription from the cognate long terminal repeat (LTR) . Bel1 can also efficiently activate the human immunodeficiency virus type 1 (HIV-1) LTR . We have previously shown that the amino-terminal 227-residue region (minimal activator region) of Bel1 can activate the HSRV LTR at low levels and that two distinct domains within the carboxy-terminal 73 residues, from residues 255 to 266 and 272 to 300, that bear little sequence homology can independently enhance the activity of the minimal activator domain (L . K . Venkatesh, C . Yang, P . A . Theodorakis, and G . Chinnadurai, J . Virol . 67:161-169, 1993) . We now report on the further characterization of these two transcriptional enhancement regions . Mutational analysis of the region comprising residues 255 to 266 indicates that a cluster of leucine residues is critical to the function of this region . Also, residues 273 to 287, which are identical in sequence to a 15-amino-acid segment near the carboxy terminus of the simian foamy virus transcriptional activator Taf, can independently enhance the activity of the minimal activator region . To delineate the region(s) of Bel1 that could function autonomously as an activator domain, we tested the activity of chimeric proteins comprising either wild-type or functionally defective forms of Bel1 fused to the DNA binding domain, Gal4(1-147), of the yeast transcriptional activator Gal4 on a synthetic promoter comprising Gal4 DNA binding sites linked to the adenovirus E1B TATA box (minimal promoter) . Gal4-Bel1 was found to activate basal transcription from the E1B TATA box at least 35-fold, and the region responsible for this activation function was localized to the carboxy-terminal 73 amino acids . When the transcriptional enhancement regions were tested for autonomous activator function as Gal4(1-147) chimeras, residues 272 to 300, but not 255 to 266, were found to activate transcription efficiently when targeted to the E1B TATA motif and also to HSRV and HIV-1 LTRs . The highly conserved region between amino acids 273 and 287 alone was found to activate transcription efficiently when targeted to the HSRV LTR but not to the E1B TATA box or the HIV-1 LTR . Thus, our results demonstrate that the carboxy-terminal 29-amino-acid region (residues 272 to 300) contributes to Bel1 transactivation by functioning as an autonomous activator of TATA motif-directed transcription in a manner similar to that of other modular transcriptional activators.(ABSTRACT TRUNCATED AT 400 WORDS)

Hum Mol Genet, 1993 Jul, 2(7), 947 - 52
The genes for X-linked ocular albinism (OA1) and microphthalmia with linear skin defects (MLS): cloning and characterization of the critical regions; Wapenaar MC et al.; We have used cell lines from patients with deletions and translocations involving the Xp22 region to map the genes for two X-linked disorders, ocular albinism type 1 (OA1) and microphthalmia with linear skin defects (MLS) . Using existing and newly isolated DNA markers, the map position within Xp22 of key patient breakpoints, defining the boundaries of the genomic regions involved in these disorders (the critical regions), has been precisely determined . A 2.6 Mb yeast artificial chromosome (YAC) contig, spanning the critical regions for these two disorders, was assembled . Detailed long-range restriction analysis of the contig established the sizes of the critical regions to be 200 kb for OA1 and 800 - 925 kb for MLS . Ten potential CpG-islands, representing candidate sites for genes, have been mapped within the 2.6 Mb region . Our data should greatly facilitate efforts aimed at cloning the genes for these developmental defects.

Hum Mol Genet, 1993 Jul, 2(7), 889 - 99
Construction of cosmid contigs and high-resolution restriction mapping of the Huntington disease region of human chromosome 4; Zuo J et al.; The gene responsible for Huntington disease (HD) has been localized to a 2.2 million base pair (Mbp) region between the loci D4S10 and D4S98 on the short arm of human chromosome 4 . As part of a strategy originally designed to clone the gene based on its chromosomal location, we and others previously identified overlapping yeast artificial chromosome (YAC) clones covering most of this region . While these YAC clones were useful for initially obtaining long-range clone continuity, a number of features of the YACs indicated that smaller clones are generally more useful in the subsequent steps of the positional cloning strategy . In this paper, we use these YAC clones to generate sets of overlapping cosmid clones covering most of the HD region . We isolated a large number of cosmids by screening a chromosome 4-specific cosmid library with labeled DNA from a minimal overlapping set of YAC clones . These cosmid clones were further analyzed by restriction mapping and hybridization experiments, leading to the assembly of 185 cosmids into eleven contigs covering more than 1.65 Mbp and to a fine-structure restriction map of the region . Nine of these contigs cover 90 percent of the 1.7 Mbp subregion between loci D4S125 and D4S98 where the HD gene is now known to lie . The detailed restriction map and the cosmid clones should facilitate the identification and localization of cDNAs and polymorphic markers, and they provide reagents for large scale DNA sequencing of this region of the human genome . Our results suggest that this strategy should be generally useful for converting YAC clones into cosmid contigs and generating high-resolution restriction maps of genomic regions of interest.

Nat Genet, 1993 Jul, 4(3), 221 - 6
Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1; Orr HT et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant disorder characterized by neurodegeneration of the cerebellum, spinal cord and brainstem . A 1.2-Megabase stretch of DNA from the short arm of chromosome 6 containing the SCA1 locus was isolated in a yeast artificial chromosome contig and subcloned into cosmids . A highly polymorphic CAG repeat was identified in this region and was found to be unstable and expanded in individuals with SCA1 . There is a direct correlation between the size of the (CAG)n repeat expansion and the age-of-onset of SCA1, with larger alleles occurring in juvenile cases . We also show that the repeat is present in a 10 kilobase mRNA transcript . SCA1 is therefore the fifth genetic disorder to display a mutational mechanism involving an unstable trinucleotide repeat.

Teratology, 1993 Jul, 48(1), 45 - 51
The effect of tolbutamide on rat embryonic development in vitro; Ziegler MH et al.; Tolbutamide (TOLB) is a sulfonylurea used to treat non-insulin-dependent diabetes mellitus and is a suspected teratogen . However, it is not possible to discriminate between potential teratogenic effects of TOLB and malformations produced by either drug-induced hypoglycemia or the diabetic state itself . We examined the direct effect of TOLB on rat embryos cultured in a rodent whole embryo culture system . CD strain rat embryos were cultured for 48 h beginning on day 9 of gestation (plug day = day 0) . Tolbutamide was added at various concentrations (90-3,600 microM) . At the end of culture, viable embryos were examined for morphological score, number of somite pairs, crown-rump and head lengths, and DNA and protein content . Tolbutamide produced dose-related decreases in all endpoints at concentrations (2,250-3,600 microM) which are two to four times the human therapeutic concentration . Sera from TOLB-treated rats were adjusted to contain equal concentrations of glucose and insulin and then used for embryo culture . Serum from TOLB-treated rats had no observable effect on embryonic development . The mechanism for the embryotoxic effect of TOLB is unknown; however, the drug was previously demonstrated to alter activity of purified yeast glutathione reductase (GR) . Because GR may be important for normal embryonic development, the effect of TOLB on this enzyme activity in cultured rat embryos was evaluated . Tolbutamide (2,700 microM) reduced embryonic GR activity by 35-57% . These results indicate that TOLB has a direct embryotoxic effect at levels 2 to 4 times the usual therapeutic serum concentrations on developing rodent embryos which may be mediated by GR inhibition.

J Clin Microbiol, 1993 Jul, 31(7), 1804 - 10
A phaeohyphomycotic cyst and peritonitis caused by Phialemonium species and a reevaluation of its taxonomy; King D et al.; Two cases of human fungal infections caused by members of the genus Phialemonium, a genus proposed by Gams and McGinnis (1983) for fungi intermediate between the genera Acremonium and Phialophora, are presented . The first case was a phaeohyphomycotic cyst on the foot of a renal transplant recipient . The fungus was detected by direct examination and histopathology and was recovered by several procedures over 4 months . It was flat, glabrous, and white becoming yellow with the production of a diffusible yellow pigment; it had conidiophores that were mostly solitary and lateral and terminal phialides and adelophialides with distinct collarettes producing cylindrical to curved conidia . The isolate resembled both Phialemonium dimorphosporum and Phialemonium curvatum, although its characteristics were more consistent with those of the latter . The second case was peritonitis in a renal transplant recipient . The fungus was white-to-cream colored and yeast like, but later became black with a green diffusible pigment, and produced obovoid conidia; it was easily identified as Phialemonium obovatum . Difficulties encountered in the identification and taxonomy of members of this genus highlight the need for standardized conditions, e.g., potato dextrose agar culture incubated at 24 to 25 degrees C for morphologic comparisons, to control significant variations due to culture conditions.

Genetics, 1993 Jul, 134(3), 837 - 45
Compositional heterogeneity and patterns of molecular evolution in the Drosophila genome; Carulli JP et al.; The rates and patterns of molecular evolution in many eukaryotic organisms have been shown to be influenced by the compartmentalization of their genomes into fractions of distinct base composition and mutational properties . We have examined the Drosophila genome to explore relationships between the nucleotide content of large chromosomal segments and the base composition and rate of evolution of genes within those segments . Direct determination of the G + C contents of yeast artificial chromosome clones containing inserts of Drosophila melanogaster DNA ranging from 140-340 kb revealed significant heterogeneity in base composition . The G + C content of the large segments studied ranged from 36.9% G + C for a clone containing the hunchback locus in polytene region 85, to 50.9% G + C for a clone that includes the rosy region in polytene region 87 . Unlike other organisms, however, there was no significant correlation between the base composition of large chromosomal regions and the base composition at fourfold degenerate nucleotide sites of genes encompassed within those regions . Despite the situation seen in mammals, there was also no significant association between base composition and rate of nucleotide substitution . These results suggest that nucleotide sequence evolution in Drosophila differs from that of many vertebrates and does not reflect distinct mutational biases, as a function of base composition, in different genomic regions . Significant negative correlations between codon-usage bias and rates of synonymous site divergence, however, provide strong support for an argument that selection among alternative codons may be a major contributor to variability in evolutionary rates within Drosophila genomes.

Mol Gen Genet, 1993 Jul, 240(1), 113 - 25
Promoter specificity and deletion analysis of three heat stress transcription factors of tomato; Treuter E et al.; Transient expression assays in transformed tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts were used to test the activity of three tomato heat stress transcription factors, HSF24, HSF8 and HSF30, in a trans-activation and a trans-repression assay . The results document differences between the three HSFs with respect to their response to the configuration of heat stress promoter elements (HSEs) in the reporter construct (promoter specificity) and to the stress regime used for activation . Analysis of C-terminal deletions identified acidic sequence elements with a central tryptophan residue, which are important for HSF activity control . Surprisingly, heterologous HSFs from Drosophila and human cells, but not from yeast, were also functional as heat stress-induced transcription factors in this tobacco protoplast system.

Chem Biol Interact, 1993 Jul, 88(1), 37 - 53
Metabolism and cytotoxicity of trans,trans-muconaldehyde and its derivatives: potential markers of benzene ring cleavage reactions; Goon D et al.; trans,trans-Muconaldehyde (MA) has been proposed to be a myelotoxic metabolite of benzene, although it has not been isolated from benzene administration in vivo . Since the reactivity and further metabolism of MA may preclude its isolation, we have examined the metabolism of MA by: (a) mixtures of yeast alcohol and aldehyde dehydrogenases, (b) mouse liver cytosol, and (c) isolated rat hepatocytes . In all three systems, MA was metabolized rapidly and the major stable end-product of metabolism was the hydroxy/acid (OH/COOH) derivative of MA . The major route of metabolism involved initial reduction to the hydroxy/aldehyde (OH/CHO) derivative . trans,trans-Muconic acid (COOH/COOH), which is used as a marker of benzene ring cleavage reactions in vivo, was also formed from MA albeit to a much lesser extent compared to the OH/COOH . The thiol reactivity, metabolism, and cytotoxicity of MA and its different redox forms (i.e., OH/OH, OH/CHO, COOH/CHO, COOH/COOH, OH/COOH) were also investigated . MA was found to react most rapidly with reduced glutathione (GSH) in a cell-free system and was also the most cytotoxic to rat hepatocytes . Apart from MA, only the OH/CHO demonstrated GSH-reactivity and cytotoxicity . The OH/CHO was a major initial metabolite in all three systems and, thus, could represent a less reactive but more diffusible derivative of MA . These studies define the metabolism and cytotoxicity of MA and its redox derivatives and suggest that the OH/COOH metabolite of MA may have relevance as a marker of ring cleavage reactions of benzene in vivo.

Proc Natl Acad Sci U S A, 1993 Jul 1, 90(13), 6100 - 4
Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination; Silver DP et al.; As a probe of whether RAG-1 and RAG-2 gene products activate other genes or form part of the recombinase itself, certain mutants of the RAG genes were assayed for their ability to activate variable-diversity-joining region {V(D)J} recombination in a plasmid substrate in fibroblasts . The results indicate that the N-terminal one-third of RAG-1, including a zinc-finger-like domain, and an acidic domain of RAG-2 are dispensable for activating V(D)J recombination in a fibroblast, although they contribute quantitatively . In contrast, deletion of the C-terminal segment of RAG-1, which has homology to a topoisomerase-like protein from yeast, abolished recombination activation . These results do not support the hypothesis that the RAG gene products are transcription factors and suggest the possibility that they are parts of the recombination machinery.

Am J Hum Genet, 1993 Jul, 53(1), 220 - 33
Identification of class-mu glutathione transferase genes GSTM1-GSTM5 on human chromosome 1p13; Pearson WR et al.; The GSTM1, GSTM2, GSTM3, GSTM4, and GSTM5 glutathione transferase genes have been mapped to human chromosome 1 by using locus-specific PCR primer pairs spanning exon 6, intron 6, and exon 7, as probes on DNA from human/hamster somatic cell hybrids . For GSTM1, the assignment was confirmed by Southern blot hybridization to a pair of 12.5/2.4-kb HindIII fragments . The GSTM1-specific primer pairs can be used to identify individuals carrying non-null GSTM1 alleles . The organization of these five genes was confirmed by the isolation of a yeast artificial chromosome clone (GSTM-YAC2) that contains all five genes . With this clone, the location of the GSTM1-GSTM5 gene cluster on chromosome 1 was confirmed by fluorescence in situ hybridization . Both regional assignment using the fractional length method and examination of probe signal with reference to R-banded chromosomes induced by BrdU places the gene cluster in or near the 1p13.3 region . The close physical proximity of the GSTM1 and GSTM2 loci, which share 99% nucleotide sequence identity over 460 nucleotides of 3'-untranslated mRNA, suggests that the GSTM1-null allele may result from unequal crossing-over.

J Exp Med, 1993 Jul 1, 178(1), 49 - 62
Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex; Fritzler MJ et al.; Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library . Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis . Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts . The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160 . The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids . The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively . The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum . Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160 . Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73 . A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin . SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide . Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

Ann Hum Genet, 1993 Jul, 57 ( Pt 3), 169 - 78
Germline rearrangement of MCC and APC detected by pulsed field gel electrophoresis and fluorescent in situ hybridization; Gayther S et al.; Pulsed field gel electrophoresis was carried out on lymphocyte DNA obtained from ten patients with the inherited premalignant condition adenomatous polyposis coli and a similar number of control samples to search for large scale molecular rearrangements in the vicinity of the APC locus . DNA from one patient showed a rearranged pattern which was interpreted as evidence for an inversion of approximately 150 kb, changing the transcriptional orientation of the neighbouring MCC gene and bringing it closer to APC . Such an interpretation was supported by fluorescent in situ hybridization data.

Zhonghua Yan Ke Za Zhi, 1993 Jul, 29(4), 233 - 5
{A study on RBC immunofunction in experimental traumatic uveitis}; Hou M; Examination of the RBC immunofunction in rabbit experimental traumatic uveitis with Guo's method of complement-labelled yeast revealed that the percentage of RBC C3b receptor rosette and the activity of red cell immune adherence (RCIA) enhancing factor in sera were markedly lower, while the percentage of RBC surface immune complex rosette, the activity of the RCIA-inhibiting factor and the CIC in sera were higher than those in the normal controls, indicating that the lowered RBC immunofunction was involved in pathogenesis of the experimental traumatic uveitis.

Somat Cell Mol Genet, 1993 Jul, 19(4), 405 - 11
Regional mapping of the gene encoding dihydroorotate dehydrogenase, an enzyme involved in UMP synthesis, electron transport, and superoxide generation, to human chromosome region 16q22; Barnes T et al.; De novo UMP synthesis is a critical metabolic pathway for nucleic acid synthesis and for a variety of metabolic pathways . The pathway is a target for many widely used cancer chemotherapy agents, several of which are pyrimidine analogs . Humans and cattle have been described with mutations in UMP synthesis that lead to serious inborn errors of metabolism . Dihydroorotate dehydrogenase (EC 1.3.3.1) (DHODH) carries out the fourth committed step in the pathway and may also be important for mitochondrial electron transport and oxygen radical metabolism . We report here that the gene encoding this enzyme in humans is located in the chromosomal region 16q22 . With the mapping of DHODH, the mapping of all the steps of UMP synthesis is complete . All three genes involved map to different human chromosomes . This information is important in consideration of regulation of UMP synthesis in mammals, including humans.

Lett Appl Microbiol, 1993 Jul, 17(1), 1 - 3
Characterization of soluble rifamycin oxidase from Curvularia lunata var . aeria; Banerjee UC; Curvularia lunata var . aeria was grown on yeast extract, peptone and carboxymethylcellulose (YPC) medium for the production of extracellular rifamycin oxidase . The enzyme was partially purified through a Sephadex G-75 column . The half lives of rifamycin oxidase at 30 degrees and 40 degrees C were 9 d and 100 min, respectively . The activation and deactivation energies of the partially purified enzyme, calculated from Arrhenius plots, were 5.80 and 35.10 kcal mol-1, respectively . The enzyme exhibited a Km (rifamycin B) value of 0.67 mmol l-1 and a Vmax of 11 mumol h-1.ml . Three metal ions, Fe2+, Ag+ and Hg2+, inhibited the enzyme in the 10-20 mmol l-1 metal ion concentration range . Catalytic activity was not affected by the chelating agent, EDTA.

J Biol Chem, 1993 Jun 25, 268(18), 13601 - 8
Characterization of the human interleukin-2 receptor gamma chain gene; Noguchi M et al.; The interleukin-2 (IL-2) receptor gamma chain is an essential component of high and intermediate affinity IL-2 receptors (IL-2Rs), playing critical roles for ligand binding and internalization . We report here the isolation and characterization of the genomic locus for human IL-2R gamma, which, like IL-2R beta, is a member of the cytokine receptor superfamily . The IL-2R gamma gene is composed of eight exons and seven introns and spans approximately 4.2 kilobases . Analogous to the IL-2R beta gene, the two pairs of conserved cysteines typical of cytokine receptor superfamily proteins are located in adjacent exons, and the conserved WSXWS motif is located in the exon preceding the one that encodes the transmembrane domain and a small part of the cytoplasmic domain . In each gene, the remainder of the cytoplasmic domain is encoded by the final two exons . Southern blot analysis suggests that IL-2R gamma is encoded by a single copy gene . Cross-hybridizing sequences were detected in DNA derived from a number of other mammalian species but not from yeast . Primer extension analysis and ribonuclease protection assays revealed that there are three principal transcription initiation sites located 32-38 nucleotides 5' to the translation initiation AUG codon . These sites are upstream of the 5' end of the published IL-2R gamma cDNA sequence . The region 5' to the transcription initiation sites exhibited promoter activity when cloned upstream of the luciferase reporter gene . With this study, the organization of the genes encoding all three chains (alpha, beta, and gamma) of the IL-2 receptor has been determined and promoters for each identified.

J Biol Chem, 1993 Jun 25, 268(18), 13422 - 33
Purification and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas; Mitsui K et al.; Calmodulin-dependent protein kinase III (CaM kinase III) phosphorylates and thereby inactivates eukaryotic elongation factor-2 (EF-2) . This enzyme, purified to homogeneity from either rabbit reticulocytes or rat pancreas, had similar properties: it migrated with an apparent M(r) of 140,000 by gel filtration, was comprised of a major polypeptide of M(r) 95,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a high affinity for CaM (half-maximal activation < 1 nM) . The M(r) 95,000 polypeptide was autophosphorylated by an intramolecular mechanism on seryl residues in the presence of Ca2+, CaM, and ATP, and phosphopeptide mapping indicated that several sites were phosphorylated . Autophosphorylation of CaM kinase III resulted in the generation of a partially Ca2+/calmodulin-independent activity . The enzyme could also be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase . Amino acid sequencing of CaM kinase III indicated that it is distinct from other known proteins including the heat-shock protein hsp90, which was recently suggested to be identical to CaM kinase III (Nygard, O., Nilsson, A., Carlberg, U., Nilsson, L., and Amons, R . (1991) J . Biol . Chem . 266, 16425-16430) . Furthermore, hsp90 did not copurify with CaM kinase III, and the M(r) 95,000 protein did not cross-react with antibodies to hsp90 . CaM kinase III exhibited Michaelis-Menten kinetics toward its substrates ATP and EF-2, with Km values of 15 and 2 microM, respectively . CaM kinase III was able to phosphorylate yeast EF-2 with an Km of 2 microM, but the enzyme did not significantly phosphorylate a variety of other substrates, confirming its identification as a novel protein kinase.

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