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 h