S. cerevisiae

Biochemistry, 1995 Apr 18, 34(15), 5011 - 7
Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA; Shivji MK et al.; In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment . Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA) . To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions . Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch . DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles . The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products . DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles . Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C . A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.

FEBS Lett, 1995 Apr 17, 363(1-2), 132 - 6
Nuclear recruitment of A1p145 subunit of replication factor C in the early G1 phase of the cell cycle in Faza 567 hepatoma cell line and hepatocyte primary cultures; Levavasseur F et al.; Using a combination of immunoprecipitation and Western blotting with Faza 567 hepatoma cell extracts revealed that the large subunit of replication factor C (A1p145; mRFC140) was in a complex with proliferating cell nuclear antigen (PCNA) . Western blotting showed that A1p145 was more abundant in nuclear extracts from butyrate-treated hepatoma cells which blocks the cells in the G1 phase of the cell cycle than from routinely cultured cells . Indirect immunoperoxidase analysis of G1 blocked Faza hepatoma cells localized A1p145 protein predominantly in the nucleoli . When hepatoma cells were stimulated to progress toward the S phase, A1p145 protein was then observed in both the cytoplasm and the nucleoplasm of these cells . Studies with early cultured normal hepatocytes which are progressing from G0 towards G1, also showed a nucleolus distribution for A1p145 . This is the first demonstration in mammalian cells that the large subunit of replication factor C is associated with PCNA in the nucleus and that its distribution within cells changes during the cell cycle.

Yeast, 1995 Apr 15, 11(4), 343 - 53
Plasmid reorganization during integrative transformation in Hansenula polymorpha; Bogdanova AI et al.; During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H . polymorpha were frequently unstable and lost plasmids while growing on non-selective medium . These transformants possessed reorganized plasmids capable of replication in H . polymorpha . Two such plasmids were isolated and characterized . It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation . Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H . polymorpha DNA . The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions . These chromosomal DNA segments apparently carried H . polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H . polymorpha with high efficiency and were maintained in transformants in an autonomous state . Sequence analysis of one such captured chromosomal fragment revealed several eight- to ten-base AT-rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al . (1986) . Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H . polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.

Genes Dev, 1995 Apr 15, 9(8), 956 - 71
The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology; Thacker C et al.; Many secreted proteins are excised from inactive proproteins by cleavage at pairs of basic residues . Recent studies have identified several serine endoproteases that catalyze this cleavage in the secretory pathways of yeast and metazoans . These enzymes belong to the kex2/subtilisin-like family of proprotein convertases . In this paper we describe the molecular characterization of the bli-4 gene from Caenorhabditis elegans, which was shown previously by genetic analysis of lethal mutants to be essential for the normal development of this organism . Sequencing of cDNA and genomic clones has revealed that bli-4 encodes gene products related to the kex2/subtilisin-like family of proprotein convertases . Analysis of bli-4 cDNAs has predicted four protein products, which we have designated blisterases A, B, C, and D . These protein products share a common amino terminus, but differ at the carboxyl termini, and are most likely produced from alternatively spliced transcripts . We have determined the molecular lesions for three bli-4 alleles (h199, h1010, and q508) that result in developmental arrest during late embryogenesis . In each case, the molecular lesions are within exons common to all of the BLI-4 isoforms . The original defining allele of bli-4, e937, is completely viable yet exhibits blistering of the adult cuticle . Molecular analysis of this allele revealed a deletion that removes exon 13, which is unique to blisterase A . No RNA transcript corresponding to exon 13 is detectable in the blistered mutants . These findings suggest that blisterase A is required for the normal function of the adult cuticle.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes Dev, 1995 Apr 15, 9(8), 897 - 910
Association of an activator with an RNA polymerase II holoenzyme; Hengartner CJ et al.; RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins . The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme . We report here the isolation and characterization of additional SRB genes . We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation . The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme . Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme . Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column . These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.

Blood, 1995 Apr 15, 85(8), 2194 - 201
Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils . Dynamics of intracellular Ca2+ stores; Theler JM et al.; The subcellular pattern of cytosolic free Ca2+ ({Ca2+}i) changes in human polymorphonuclear neutrophils (PMNs) was studied using imaging of fura-2 fluorescence (time resolution 12.5 ratios/s) to determine whether PMNs could obtain directional information from the {Ca2+}i signal . {Ca2+}i changes were observed during initial adherence, the subsequent chemotactic movement, and the phagocytosis of opsonized yeast particles . Initial adherence was followed by a rapid increase in {Ca2+}i (from 90 +/- 10 to 290 +/- 40 nmol/L in 6.5 +/- 2.5 seconds; +/- SEM, n = 10), apparently homogeneously distributed over the entire cytoplasm, which preceded the spreading of the PMNs . {Ca2+}i increases after the contact of the PMNs with yeast particles were of lower mean amplitude; {Ca2+}i increased simultaneously throughout the cytosol . In the absence of extracellular Ca2+, multiple phagocytotic events could proceed normally without a mandatory {Ca2+}i transient . In PMNs polarized on phagocytosis, gradients in {Ca2+}i could be observed . {Ca2+}i was more elevated in the periphagosomal area than in the remaining parts . Taken together, these data show that {Ca2+}i waves do not provide the neutrophil with directional information during chemotaxis and phagocytosis . Sustained small inhomogeneity of {Ca2+}i levels are consistent with a proposed redistribution of releasable Ca2+ stores on phagocytosis.

J Biol Chem, 1995 Apr 14, 270(15), 8837 - 43
The retinoblastoma susceptibility gene product represses transcription when directly bound to the promoter; Adnane J et al.; Rb represses E2F-mediated transcription in part by blocking the trans-activation domain of E2F . In addition, Rb can convert an E2F binding site from a positive to a negative element . To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4 . Here, we report that GAL4-Rb can repress transcription mediated by either Sp1, AP-1, or p53, dependent upon the presence of both the GAL4 DNA binding domain and GAL4 binding sites . Moreover, GAL4-Rb inhibited the activity of the herpes simplex virus tk promoter from GAL4 binding sites located at a distance from the promoter . In contrast, GAL4-Rb was unable to repress basal transcription . Cotransfection of specific cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the repressive activity of GAL4-Rb . The domains of Rb involved in mediating the repression of transcription were mapped to regions that are overlapping, but not identical, to those required for the interaction with E2F . We propose that Rb can function as a general repressor of transcription when bound to the promoter region.

Nature, 1995 Apr 13, 374(6523), 657 - 60
Basal promoter elements as a selective determinant of transcriptional activator function; Das G et al.; In eukaryotes, activation of transcription involves an interplay between activators bound to cis-regulatory elements and factors bound to basal elements near the start site of transcription . The basal elements, for example the TATA box or proximal sequence element (PSE) of small nuclear RNA (snRNA) promoters, nucleate the assembly of basal transcription complexes, components of which interact with activators . Although one basal transcription complex can interact with many activators, it is unclear whether different basal transcription complexes can direct different responses to particular activators . We show here that changing the arrangement of basal elements can alter the response to transcriptional activation domains . Indeed, in the human U6 snRNA promoter, point mutation of either a TATA box or PSE results in diametrically opposed responses to VP16- and Sp1-derived activation domains . These basal elements can even discriminate small changes in an activation domain . Thus the arrangement of basal promoter elements provides a mechanism for differential regulation of transcription.

Proc Natl Acad Sci U S A, 1995 Apr 11, 92(8), 3171 - 4
Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells; Peterson SR et al.; The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350) . The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5 . Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells . The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme . In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination . A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity . These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

Cell, 1995 Apr 7, 81(1), 73 - 83
Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis; Kamper J et al.; In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus . The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides . Only combinations of bE and bW of different allelic origin are active . We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon . By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles . Dimerization involves the N-terminal variable domains . Different point mutants of bE2 were isolated that function in combination with bW2 . The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system . Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins . Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

J Biol Chem, 1995 Apr 7, 270(14), 8225 - 32
The cAMP response element binding protein synergizes with other transcription factors to mediate cAMP responsiveness; Roesler WJ et al.; The cAMP responsiveness of the promoter for phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is mediated by a synergistic interaction between a complex regulatory region, which binds liver-enriched transcription factors, and a typical cAMP response element (CRE) . Although a role for the CRE-binding protein (CREB) in the cAMP-responsiveness of this promoter has been generally assumed, some uncertainty remains due to the observations that several C/EBP-related proteins bind with near equal affinity, relative to CREB, to this particular CRE . Thus, a detailed analysis of the involvement of CREB in this synergism was undertaken in HepG2 cells . Gel mobility shift assays demonstrate that a CRE probe is bound by CREB present in HepG2 cells . Furthermore, we show that a dominant repressor of CREB is able to significantly reduce the cAMP responsiveness of the PEPCK promoter in HepG2 cells . Finally, we demonstrate using a GAL4-CREB fusion protein that CREB is able to synergize with the liver-enriched factors bound upstream on the PEPCK promoter to mediate a liver-specific response to cAMP . Examination of several mutant forms of CREB allow us to conclude that the "synergy" domain of CREB resides within amino acid residues 83-203, and that residues 83-145 can mediate a partial synergistic response . This study establishes that CREB is able to synergize with liver-enriched transcription factors to mediate a tissue-specific response to cAMP.

Cell, 1995 Apr 7, 81(1), 53 - 62
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia; Nobes CD et al.; Rho and rac, two members of the ras-related superfamily of small GTPases, regulate the polymerization of actin to produce stress fibers and lamellipodia, respectively . We report here that cdc42, another member of the rho family, triggers the formation of a third type of actin-based structure found at the cell periphery, filopodia . In addition to stress fibers, rho controls the assembly of focal adhesion complexes . We now show that rac and cdc42 also stimulate the assembly of multimolecular focal complexes at the plasma membrane . These complexes, which are associated with lamellipodia and filopodia, contain vinculin, paxillin, and focal adhesion kinase, but are distinct from and formed independently of rho-induced focal adhesions . Activation of cdc42 in Swiss 3T3 cells leads to the sequential activation of rac and then rho, suggesting a molecular model for the coordinated control of cell motility by members of the rho family of GTPases.

Oncogene, 1995 Apr 6, 10(7), 1301 - 6
Functional diversity of LIM proteins: amino-terminal activation domains in the oncogenic proteins RBTN1 and RBTN2; Sanchez-Garcia I et al.; The RBTN1 and RBTN2 genes are activated by distinct translocations involving chromosome 11 in some T cell acute leukaemias . The RBTN proteins belong to the LIM family which comprises proteins with one, two or three cysteine-rich LIM domains, sometimes together with homeodomains or protein kinase domains . The RBTN1 and RBTN2 proteins comprise only tandem LIM domains . We report that RBTN1 and RBTN2 proteins are capable of supporting transcriptional transactivation of specific reporter genes in transfection assays . The results, using intact proteins or fusions with the homeodomain of the heterologous protein Isl-1, show that this transcriptional activation ability resides in the NH2-terminal parts of both proteins . The use of yeast assays with RBTN2 shows that RBTN2 forms homodimers and that the NH2-terminal 27 amino acids are sufficient to facilitate transcriptional transactivation . These data expand the functional diversity of the LIM-domain protein family and they augment the previously defined relationship between chromosomal translocations and transcriptional activation.

Nature, 1995 Apr 6, 374(6522), 562 - 5
A role for retinoblastoma protein in potentiating transcriptional activation by the glucocorticoid receptor; Singh P et al.; The Saccharomyces cerevisiae SNF2/SWI2 protein is essential for the regulated expression of a variety of genes . A human SWI2/SNF2 homologue, hBrm, is a positive participant in glucocorticoid-receptor-mediated transcription, but its mechanism of action is not known . The retinoblastoma protein, RB, has also been shown to stimulate the transcription of several genes, although the target for RB has not been identified in any of these transcriptional events . Here we show that RB upregulates glucocorticoid-receptor-mediated transcription . The effect of either RB or hBrm is dependent on the presence of the other . Furthermore, we demonstrate that RB and hBrm interact with one another in vitro and in vivo . These results highlight a new role for RB, which is to interact with hBrm in order to potentiate glucocorticoid-receptor-activated transcription.

EMBO J, 1995 Apr 3, 14(7), 1490 - 7
TBP mutants defective in activated transcription in vivo; Arndt KM et al.; The TATA box binding protein (TBP) plays a central and essential role in transcription initiation . At TATA box-containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex . Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro . To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription . One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source . These phenotypes are due to promoter-specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element . The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo.

EMBO J, 1995 Apr 3, 14(7), 1349 - 59
A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting; Gruhler A et al.; Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence . In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate . Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed . This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX . Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane . This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins . These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus . This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix . The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.

EMBO J, 1995 Apr 3, 14(7), 1329 - 39
The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi; Schimmoller F et al.; Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex . A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected . Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization . Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction . We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.

Mol Cell Biol, 1995 Apr, 15(4), 2304 - 10
AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation; Datta K et al.; The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain) . The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain) . In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes . The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2 . The AH/PH domain-mediated interactions depend on the integrity of the entire domain . Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt . To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells . Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

Mol Cell Biol, 1995 Apr, 15(4), 1953 - 60
Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence; Nandabalan K et al.; The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth . Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells . Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation . The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes . The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency . The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA . Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate . RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron . Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.

Mol Cell Biol, 1995 Apr, 15(4), 1835 - 46
Activation of CLN1 and CLN2 G1 cyclin gene expression by BCK2; Di Como CJ et al.; The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6 . We isolated 12 complementation groups of mutants that require CLN3 . The members of one of these complementation groups have mutations in the BCK2 gene . In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA . In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA . The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2 . Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs . The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1.

J Virol, 1995 Apr, 69(4), 2605 - 10
Delineating minimal protein domains and promoter elements for transcriptional activation by lentivirus Tat proteins; Southgate CD et al.; Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication . In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action . We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts . In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multiple binding sites for the cellular transcription factor SP1 . We delineate a 114-amino-acid region of the SP1 glutamine-rich activation domain that when fused to the GAL4 DNA binding domain can support transcription activation by Tat . Using these Tat and SP1 derivatives, we show that Tat activation can be reconstructed on a completely synthetic promoter lacking all cis-acting elements unique to the human immunodeficiency virus long terminal repeat . Our results indicate that lentivirus Tat proteins have essential properties of typical cellular transcriptional activators and define useful reagents for studying the detailed mechanism of Tat action.

Genetics, 1995 Apr, 139(4), 1701 - 9
A genetic analysis of the 63E-64A genomic region of Drosophila melanogaster: identification of mutations in a replication factor C subunit; Harrison SD et al.; We have performed an F2 genetic screen to identify lethal mutations within the 63E-64A genomic region . We have isolated 122 mutations in 20 different complementation groups . Of these groups, 16 are represented by multiple alleles . We have also established that the Rop and Ras2 genes are located within the 63E-64A genomic domain at 64A10,11 . We have sequenced 10.2 kb of DNA surrounding this gene pair and find that in addition to Rop and Ras2 there is another gene located within this DNA sequence . The gene product, which we have named Rfc40, shows 68% identity to the 40-kDa subunit of replication factor C . We find that the members of one complementation group (13 alleles) derived from our screen correspond to mutations in the Rop gene, whereas the members of another (five alleles) correspond to mutations in the Rfc40 gene . In addition we have isolated 11 new mutant alleles of the disembodied gene.

Plant Mol Biol, 1995 Apr, 28(1), 195 - 201
The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression; Liu Z et al.; Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition . All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect . The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect . This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein . Small deletions at either the 5' or 3' end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect . A 36 bp region at the 5' end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences . Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not . These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.

Plant Physiol, 1995 Apr, 107(4), 1217 - 23
Molecular features and mitochondrial import pathway of the 14-kilodalton subunit of cytochrome c reductase from potato; Braun HP et al.; The cytochrome c reductase complexes from fungi and mammals both contain a 14-kD protein (yeast, 14.4 kD; bovine, 13.4 kD) that does not directly participate in electron transfer but possibly is indirectly involved in the function of the complex and has a role in assembly of the multimeric enzyme . A subunit of comparable size was identified for the bc1 complex of higher plants . The 14-kD protein from potato (Solanum tuberosum) was specifically separated from the isolated protein complex in the presence of 6 M urea and is, therefore, assumed to be a peripheral component . Direct sequence analysis of the proteins from potato and wheat (Triticum aestivum) and isolation of corresponding cDNA clones for the subunit from potato revealed clear similarity to the equivalent proteins from yeast and bovine . The wheat 14-kD protein seems to occur in two isoforms . The 14-kD protein from plants is very hydrophilic, has a characteristic charge distribution, and contains no potential membrane-spanning helices . In vitro import of the radiolabeled 14-kD protein from potato into isolated mitochondria depends on the membrane potential across the inner mitochondrial membrane . The protein seems to lack a cleavable mitochondrial presequence, because it is not processed upon translocation . Possible intramolecular regions involved in targeting of the 14-kD protein to plant mitochondria are discussed.

Eur J Biochem, 1995 Apr 1, 229(1), 1 - 13
The MADS-box family of transcription factors; Shore P et al.; The MADS-box family of transcription factors has been defined on the basis of primary sequence similarity amongst numerous proteins from a diverse range of eukaryotic organisms including yeasts, plants, insects, amphibians and mammals . The MADS-box is a conserved motif found within the DNA-binding domains of these proteins and the name refers to four of the originally identified members: MCM1, AG, DEFA and SRF . Several proteins within this family have significant biological roles . For example, the human serum-response factor (SRF) is involved in co-ordinating transcription of the protooncogene c-fos, whilst MCM1 is central to the transcriptional control of cell-type specific genes and the pheromone response in the yeast Saccharomyces cerevisiae . The RSRF/MEF2 proteins comprise a sub-family of this class of transcription factors which are key components in muscle-specific gene regulation . Moreover, in plants, MADS-box proteins such as AG, DEFA and GLO play fundamental roles during flower development . The MADS-box is a contiguous conserved sequence of 56 amino acids, of which 9 are identical in all family members described so far . Several members have been shown to form dimers and consequently two functional regions within the MADS-box have been defined . The N-terminal half is the major determinant of DNA-binding specificity whilst the C-terminal half is necessary for dimerisation . This organisation allows the potential formation of numerous proteins, with subtly different DNA-binding specificities, from a limited number of genes by heterodimerisation between different MADS-box proteins . The majority of MADS-box proteins bind similar sites based on the consensus sequence CC(A/T)6GG although each protein apparently possesses a distinct binding specificity . Moreover, several MADS-box proteins specifically recruit other transcription factors into multi-component regulatory complexes . Such interactions with other proteins appears to be a common theme within this family and play a pivotal role in the regulation of target genes.

Dev Biol, 1995 Apr, 168(2), 613 - 26
Molecular and genetic analysis of the Drosophila mas-1 (mannosidase-1) gene which encodes a glycoprotein processing alpha 1,2-mannosidase; Kerscher S et al.; Glycosylation is an important mechanism for modulating the physicochemical and biological properties of proteins in a stage- and tissue-specific manner . The enzymology of this process is just beginning to be understood . Here we present the molecular analysis of mas-1 (mannosidase-1), a Drosophila gene with significant homologies to mammalian and Saccharomyces cerevisiae glycoprotein processing alpha 1,2-mannosidases . An enhancer-trap P-element inserted upstream of mas-1 leads to highly specific lacZ expression in the lobula plate giant neurons, cells that mediate the large-field optomotor response . This staining, however, seems to reflect only a small part of the complex expression pattern of the mas-1 gene: Two promoters produce alternative transcripts that show individual spatial distributions during embryonic development, including a maternal contribution . Both transcripts code for type II transmembrane proteins which differ in their N-terminal parts . Null mutants in mas-1 display defects in the embryonic PNS, in the wing, and in the adult eye . These findings illustrate that the processing of N-linked glycans plays a functional role in Drosophila development . There is, however, ample evidence for genetic and biochemical redundancy in the mannose-trimming steps of this pathway.

J Cell Biol, 1995 Apr, 129(2), 345 - 55
Pmp27 promotes peroxisomal proliferation; Marshall PA et al.; Peroxisomes perform many essential functions in eukaryotic cells . The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes . This process is not understood at the molecular level . Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate . This growth substrate is metabolized by peroxisomal enzymes . We have identified a protein, Pmp27, that promotes peroxisomal proliferation . This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced . Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii . Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer . Its expression is regulated by oleate . The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid . The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type . Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation . However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes . In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes . We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.

Genes Dev, 1995 Apr 1, 9(7), 821 - 31
Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters; Tzamarias D et al.; The yeast Cyc8(Ssn6)-Tup1 complex is required for transcriptional repression of distinct sets of genes that are regulated by glucose, oxygen, cell type, and DNA damage . It has been proposed that the Cyc8-Tup1 complex is a corepressor that is recruited to promoters by interacting with pathway-specific DNA-binding proteins . Previously, we showed that a specific region of Tup1 mediates the general transcriptional repression function of the complex . Here, we define functional domains of Cyc8, a protein consisting primarily of 10 tandem copies of a TPR motif . Distinct combinations of TPR motifs are required specifically for direct interaction with Tup1, repression of oxygen-regulated genes, and repression of glucose-regulated genes . In contrast, the WD motifs of Tup1 are not essential for repression of genes regulated by glucose and oxygen, but they are required for those regulated by cell type and DNA damage . In addition, we show that the Cyc8-Tup1 complex functions both as a corepressor and an inhibitor of Mig1, a protein that binds to promoters of glucose-repressible genes . These observations suggest that different Cyc8 TPR motifs and the Tup1 WD domain mediate distinct protein-protein interactions that link the Cyc8-Tup1 corepressor to structurally dissimilar DNA-binding proteins required for pathway-specific regulation.

J Cell Biol, 1995 Apr, 129(1), 25 - 34
Mas37p, a novel receptor subunit for protein import into mitochondria; Gratzer S et al.; By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria . Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions . Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration-driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p . Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p . Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other . We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.

J Cell Biol, 1995 Apr, 129(1), 179 - 88
A talin homologue of Dictyostelium rapidly assembles at the leading edge of cells in response to chemoattractant; Kreitmeier M et al.; In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved in the control of cell motility and chemotaxis, monoclonal antibodies were raised against proteins that had been enriched on an F-actin affinity matrix . One antibody recognized a protein distinguished by its strong accumulation at the tips of filopods . These cell-surface extensions containing a core of bundled actin filaments are rapidly protruded and retracted by cells in the growth-phase stage . The protein of 269 kD turned out to resemble mouse fibroblast talin (Rees et al., 1990) in its primary structure . The fit is best among the first 400-amino acid residues of the NH2-terminal region where identity between the two proteins is 44% and the last 200-amino acid residues of the COOH-terminal region with 36% identity . In the elongated cells of the aggregation stage the Dictyostelium talin is accumulated at the entire front where also F-actin is enriched . Since this protein exists in a soluble state in the cytoplasm, mechanisms are predicted that cause accumulation at sites of the cell where a front is established . Evidence for receptor-mediated accumulation was obtained by local stimulation of cells with cAMP . When a new front was induced by the chemoattractant, the talin accumulated there within half a minute, indicating a signal cascade in Dictyostelium responsible for assembly of the talin beneath sites of the plasma membrane where chemoattractant receptors are strongly activated . The ordered assembly of the talin homologue together with actin and a series of other proteins is considered to play a key role in chemotactic orientation.

Exp Cell Res, 1995 Apr, 217(2), 477 - 83
The growth-inhibitory block of TGF-beta is located close to the G1/S border in the cell cycle; Kletsas D et al.; Transforming growth factor-beta (TGF-beta) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF) . The inhibition of the PDGF-BB action by TGF-beta was independent of the induction of mRNAs for the PDGF-A chain and PDGF-beta receptor, the predominant types of PDGF receptor in human fibroblasts . The TGF-beta-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (GO) state to the S phase of the cell cycle . Indeed, TGF-beta upregulated the "early" genes c-myc, c-fos, and junB and downregulated the growth arrest-specific (gas) genes . These results suggest that the inhibition of DNA synthesis by TGF-beta in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-beta block comparatively late in the GO to S transition . In cultures of senescent human fibroblasts TGF-beta stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.

Curr Biol, 1995 Apr 1, 5(4), 376 - 9
Cell-cycle checkpoints . Keeping mitosis in check; Humphrey T et al.; Mutations in an essential yeast gene, encoding DNA polymerase epsilon, abolish the dependence of mitosis on the completion of DNA replication, suggesting that the replication complex provides the checkpoint signal.

J Cell Sci, 1995 Apr, 108 ( Pt 4), 1541 - 52
Localization of the small GTP-binding protein rab1p to early compartments of the secretory pathway; Saraste J et al.; We have studied the localization of the small GTPase rab1p in different cell types using polyclonal antibodies prepared against the rab1A isoform of the protein . Immunofluorescence microscopy of normal rat kidney (NRK) and mouse myeloma cells showed the association of the protein with the Golgi complex and peripheral sites where it colocalized with p58, a pre- and cis-Golgi marker protein . Rab1p and p58 also had similar distributions in membrane fractions derived from rat pancreas microsomes . Both were concentrated in two intermediate density subfractions between the rough endoplasmic reticulum and trans-Golgi, whereas rab6p, previously localized to middle and trans-Golgi, was enriched in the light density trans-Golgi fraction . Immunoperoxidase electron microscopy of NRK and myeloma cells revealed the association of rab1p with 1-2 cisternae, vacuolar, and tubulovesicular membranes in the cis-Golgi region . The rab1p-specific staining typically covered the entire lateral surface of the cisternae but, in weakly stained cells, local labeling between closely opposed membranes could also be seen . The rab1p-positive pre-Golgi compartment had a predominantly tubulovesicular appearance in NRK cells whereas in myeloma cells it consisted of vacuoles surrounded by rab1p-positive vesicles and tubules of heterogeneous size . In both cell types the rough ER cisternae and the nuclear envelope contained negligible labeling and no continuities between these and the rab1p-positive membranes were observed . In addition, in myeloma cells the smooth ER subcompartment, containing endogenous retrovirus particles, was devoid of rab1p-labeling . These results indicate that the pre-Golgi (intermediate) compartment consists of different membrane domains and its morphology can vary considerably between different cell types . Further, they suggest that the recruitment of rab1p to membranes occurs predominantly in a post-ER location and that the protein functions in targeting/fusion events within the pre- and cis-Golgi membranes.

Curr Opin Genet Dev, 1995 Apr, 5(2), 174 - 9
Chromatin multiprotein complexes involved in the maintenance of transcription patterns; Orlando V et al.; In Drosophila, the maintenance of active and inactive patterns of gene expression during development involves the activity of two genetically complex systems . Molecular analysis of the components, apparently acting in large multiprotein complexes, has allowed a substantial advancement in our understanding of the role of chromatin higher order structures in gene regulation and nuclear organization . The Polycomb-group factors induce heterochromatin-like structures on genes that need to be stably and heritably inactivated . The role of the trithorax-group factors is to counteract these repressed chromatin domains and thus to render the genes accessible to activating factors.

J Biochem (Tokyo), 1995 Apr, 117(4), 888 - 96
Nucleotide sequence and characterization of the large mitochondrial rRNA gene of Penicillium urticae, and its comparison with those of other filamentous fungi; Yamamoto H et al.; The nucleotide sequence of a large rRNA gene and its flanking regions in cloned fragments of mitochondrial DNA from a patulin producer, Penicillium urticae NRRL2159A, was determined by dideoxy sequencing, and the 5' end and intron-exon border of the 1-rRNA gene were determined by primer extension analysis and RNA sequencing, respectively . In addition to the extensive sequence homology of the 3' end of the P . urticae mt 1-rRNA gene with those of Aspergillus nidulans and Neurospora crassa, the P . urticae gene had a 1,685 bp intron which separates a 3,307 bp 5' exon and a 583 bp 3' exon . In spite of being closely related Penicillium species, the size of the 5' exon of the P . urticae mt 1-rRNA is 472 bp larger than that of P . chrysogenum, whereas the sizes of the 3' exon and intron of P . urticae are very similar to those of P . chrysogenum (581 bp for the 3' exon and 1,678 bp for the intron) . The intron of P . urticae contains a structure similar to the consensus one of the self splicing group IA intron and a large open reading frame suggested to be a gene for ribosomal protein S5 . A sequence similar to the I-SceI recognition sequence was found at the exon-intron border . Extensive sequence homology was observed between P . urticae and P . chrysogenum, exceptions being in four regions in the 5' exon . These non-homologous regions were located in the hairpin and variable regions outside of the core structures . Comparison of the mt 1-rRNA sequences of several filamentous fungi revealed that the above four non-homologous regions are greatly expanded, and two other non-homologous regions appear at the 3' ends of the 5' exon and 3' exon.

J Biochem (Tokyo), 1995 Apr, 117(4), 836 - 44
Chemical cross-linking of activated coagulation factor VII with soluble tissue factor: calcium ions are not essential for full amidolytic activity of the factor VIIa-tissue factor complex after complex formation; Miyata T et al.; In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTF alpha) with a high content of mannose residues was produced in abundance and 37 kDa sTF beta was obtained in a low yield {Shigematsu et al . (1992) J . Biol . Chem . 267, 21329-21337} . To obtain sTF beta in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast . We found that the yield of the beta form of the Asn137-to-Ala mutant (designated as sTF beta NNA) was threefold higher (3 mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats . Since it has been reported that calcium ions are required for the effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity . Here, we report the enzymatic characterization of a factor VIIa-sTF beta NNA complex cross-linked with a homobifunctional reagent, bis(sulfosuccinimidyl) suberate . The factor VIIa-sTF beta NNA complex cross-linked in the presence of 5 mM calcium ions or 50 mM EDTA was purified . The cross-linked complex did not show factor X activation in the presence of phospholipids . However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Semin Cell Biol, 1995 Apr, 6(2), 89 - 94
Control of signal transduction and morphogenesis by Ras; Hughes DA; The single Ras homologue (Ras1) of S . pombe regulates two distinct processes: (1) Signal transduction through a MAP kinase-like protein kinase cascade in response to mating pheromones . In this pathway Ras1 interacts with the protein kinase Byr2 and leads to its activation in conjunction with a signal from the receptor-coupled, heterotrimeric G protein . (2) Polarized cell growth both during the cell cycle and during directed cell extension towards a mating partner . Ras1 interacts with Ral1/Scd1, a putative guanine-nucleotide-exchange factor, which could activate Cdc42, Rho-like GTP-binding protein . Cdc42 may regulate the dynamics of the actin cytoskeleton.

Cytokine, 1995 Apr, 7(3), 223 - 31
Molecular cloning and expression of DNA encoding ovine interleukin 2; Bujdoso R et al.; We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA . The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts . The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast . P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient . Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2 . Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme . After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells . The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Genes Dev, 1995 Apr 1, 9(7), 855 - 68
A novel role for a U5 snRNP protein in 3' splice site selection; Umen JG et al.; The choice of a 3' splice site in Saccharomyces cerevisiae introns involves recognition of a uridine-rich tract upstream of the AG dinucleotide splice junction . By isolating mutants that eliminate the normal preference for uridine-containing 3' splice sites in a cis-competition, we identified a mutation that is an allele of PRP8, prp8-101 . This was unexpected because previous analysis has demonstrated that the U5 snRNP protein encoded by PRP8 is required for spliceosome assembly prior to the first catalytic step of splicing . In contrast, the uridine recognition defect caused by the prp8-101 mutation selectively inhibits the second catalytic step of splicing . This defect is seen not only in 3' splice site cis-competitions but also in the splicing of an unusual intron in the TUB3 gene and in the ACT1 intron when utilization of its 3' splice site is rate limiting for splicing . Consistent with a direct role in 3' splice site selection, Prp8 can be cross-linked to the 3' splice site during the splicing reaction . These data demonstrate a novel function for Prp8 in 3' splice site recognition and utilization.

J Biol Chem, 1995 Mar 31, 270(13), 7731 - 6
Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor; Fransen M et al.; To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system . We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification . Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast . Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane . All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.

J Biol Chem, 1995 Mar 31, 270(13), 7551 - 7
Phenylalkylamine Ca2+ antagonist binding protein . Molecular cloning, tissue distribution, and heterologous expression; Hanner M et al.; We recently characterized (Moebius, F . F., Burrows, G . G., Striessnig, J., and Glossmann H . (1993) Mol . Pharmacol . 43, 139-144) and purified (Moebius, F . F., Hanner, M., Knaus, H . G., Weber, F., Striessnig, J., and Glossmann, H . (1994) J . Biol . Chem . 269, 29314-29320) a binding protein for the phenylalkylamine Ca2+ antagonist emopamil . The emopamil-binding protein (EBP) acts as a high affinity acceptor for several antiischemic drugs and thus represents a potential common molecular target for antiischemic drug action . Degenerate oligonucleotides were synthesized according to the N-terminal amino acid sequence of purified EBP and used to amplify a guinea pig cDNA with reverse transcriptase-polymerase chain reaction and to clone full-length cDNAs from guinea pig and human liver cDNA libraries . The cDNAs coded for 229 (guinea pig) and 230 (human) amino acid 27-kDa polypeptides without significant sequence homology with any known protein . However, EBP shared structural features with pro- and eukaryotic drug transport proteins . The amino acid identity between human and guinea pig EBP was 73% . Hydrophobicity plots predicted four transmembrane segments . The C terminus contained a lysine-rich consensus sequence for the retrieval of type I integral membrane proteins to the endoplasmic reticulum . The heterologous expression of human and guinea pig EBP in Saccharomyces cerevisiae demonstrated that the expression of EBP alone is sufficient to form high affinity drug- and cation-binding domains identical to the {3H}-emopamil-binding site of guinea pig liver . Northern and Western blot analysis revealed high abundance of EBP in guinea pig epithelial tissues as liver, bowel, adrenal gland, testis, ovary, and uterus and low densities in brain, cerebellum, skeletal muscle, and heart . EBP is suggested to be the first structurally characterized member of a family of high affinity microsomal drug acceptor proteins carrying so called sigma-binding sites.

J Biol Chem, 1995 Mar 31, 270(13), 7341 - 6
Identification of two transcription activation units in the N-terminal domain of the human androgen receptor; Jenster G et al.; To locate in detail the regions in the human androgen receptor (AR) involved in transcription activation, a series of N-terminal deletions was introduced in the wild type AR and in a constitutively active AR . The different constructs were tested for their capacity to activate transcription . Almost the entire N-terminal domain (residues 1-485) was necessary for full wild type AR activity when cotransfected with the (GRE)2tkCAT reporter in HeLa cells . In contrast, a smaller part of the N-terminal domain (amino acids 360-528) was sufficient for the constitutively active AR to induce transcription of the same (GRE)2tkCAT reporter in HeLa cells . This demonstrates the capacity of the AR to use different regions in the N-terminal domain as transcription activation units (TAUs) . To obtain additional information of AR N-terminal TAUs, the GAL4 DNA binding domain was linked to either the entire or parts of the AR N-terminal domain and cotransfected with the (UAS)2tkCAT reporter in HeLa cells . The results confirmed that the first 485 amino acid residues accommodate a transcription activation function . When the chimeric AR-GAL4 constructs were tested on a different reporter ((UAS)5E1bCAT), a small shift in position of the TAU, responsible for full transcription activation, was observed . The data presented show that the size and location of the active TAU in the human AR is variable, being dependent on the promoter context and the presence or absence of the ligand binding domain.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2770 - 4
A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects; Macreadie IG et al.; Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain . Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement . Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs . Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity . A protein with functions relating to the yeast cytoskeleton, Sac1p {Cleves, A . E., Novick, P.J . & Bankaitis, V.A . (1989) J . Cell Biol . 109, 2939-2950}, shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions . The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest {Levy, D.N., Fernandes, L.S., Williams, W.V . & Weiner, D.B . (1993) Cell 72, 541-550} . We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2587 - 91
A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp . strain PC-2 is highly homologous to vertebrate cyclophilin B; Chen H et al.; A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp . strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1 . The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8 . Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM . The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs . By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated . It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal . The Orpinomyces PC-2 CyP is a typical type B CyP . The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins . Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2563 - 7
A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase; Huibregtse JM et al.; E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18 . The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis . E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates . The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins . Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation . Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin . Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin . These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).

Nucleic Acids Res, 1995 Mar 25, 23(6), 1044 - 9
Analysis of CYS3 regulator function in Neurospora crassa by modification of leucine zipper dimerization specificity; Paietta JV; The CYS3 positive regulator is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of sulfur-controlled structural genes in Neurospora crassa . An approach of modifying the dimerization specificity of the CYS3 leucine zipper was used to determine whether the in vivo regulatory function of CYS3 requires the formation of homodimeric or heterodimeric complexes . Two altered versions of CYS3 with coiled coil elecrostatic interactions favorable to heterodimerization showed restoration of wild-type CYS3 function only when simultaneously expressed in a delta cys-3 strain . In addition, constructs having the CYS3 leucine zipper swapped for that of the oncoprotein Jun or the CYS3 leucine zipper extended by a heptad repeat showed wild-type CYS3 function when transformed into a delta cys-3 strain . Gel mobility shift and immunoprecipitation assays were used to confirm the modified CYS3 proteins dimerization and DNA binding properties . The studies, which precluded wild-type CYS3 dimerization, indicate that in vivo CYS3 is fully functional as a homodimer since no interaction was required with other leucine zipper proteins to activate sulfur regulatory and structural gene expression . The results demonstrate the utility of leucine zipper modification to study the in vivo function of bZIP proteins.

J Biol Chem, 1995 Mar 24, 270(12), 6966 - 74
Transcriptional regulation by p53 . Functional interactions among multiple regulatory domains; Hsu YS et al.; The tumor suppressor p53 protein possesses activities typical of eukaryotic transcriptional activators; p53 binds to specific DNA sequences and stimulates transcription of the target genes . By a series of deletion and domain-swapping studies, we report here that (i) p53 has two auxiliary domains, which have little effect on the DNA binding activity of its core domain but are capable of modulating its transactivation activity in a target site-dependent manner; (ii) p53 contains two cell-specific transcriptional inhibitory domains, I1 and I2, which are active in Saos-2 and HeLa cells but not in HepG2 and Hep3B cells; and (iii) I1 inhibits the activity of several structurally different activating regions . These results demonstrate that the apparent transcriptional activity of p53 is determined by collaborations among its regulatory domains, its target sites, and the cellular environment.

Cell, 1995 Mar 24, 80(6), 949 - 57
REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons; Chong JA et al.; Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells . We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element . Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein . REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system . We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

Mol Cell Biochem, 1995 Mar 23, 144(2), 105 - 8
Thyroid hormone inhibits fatty acid synthase gene transcription in chicken liver; Kameda K; The effect of triiodothyronine (T3) on regulation of fatty acid synthase in chicken liver was investigated . In hypothyroid animals, enzyme activity was about one half of that in euthyroid animals . T3 treatment increased the enzyme activity in hypothyroid animals . There is little difference in both the mRNA concentration and the transcription rate between euthyroid and hypothyroid animals . T3 treatment markedly decreased both the mRNA concentration and the transcription rate in euthyroid and hypothyroid animals . These results suggested that T3 maintained the normal level of enzyme expression primarily by stimulating the post-transcriptional step, while the transcription of the gene was inhibited by hyperthyroidism.

Biochemistry, 1995 Mar 21, 34(11), 3632 - 9
Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions; Lamhasni S et al.; Topoisomerase II overexpressed in yeast was purified to near homogeneity . The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological assays at different protein and salt conditions . First, sedimentation equilibrium was preferred over the other analytical ultracentifuge methods as it is based on firm theoretical grounds and does not require assumptions about the shape of the molecule . The tendency of topoisomerase II to self-associate into dimers was confirmed and shown to depend on both the enzyme concentration and the concentration of salt used . Analysis at five initial protein concentrations (from 0.08 to 1.05 mg/mL, i.e., 0.5-65 microM) provided evidence for a single monomer-dimer equilibrium characterized at 150 mM KCl and 20 degrees C by an association constant, Ka, of approximately 4.8 10(5) M-1 and a delta G degree of approximately -7.5 kcal mol-1 . Under these conditions, for a topoisomerase II concentration of 0.08 mg/mL (i.e., 0.5 microM) in the ultracentrifuge cell, almost 80% of the enzyme were found dissociated . Increase of KCl (from 80 to 400 mM) in the medium provoked a continuous change of the association equilibrium so that a value of Ka approximately 10(5) M-1 corresponding to delta G degree approximately -7 kcal mol-1 was found for topoisomerase II in 400 mM KCl at 20 degrees C . Second, circular dichroism (CD) showed the sensitivity of the topoisomerase II secondary structure to salt concentration, the observed variations being apparently dependent upon the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1995 Mar 20, 246(6), 739 - 44
RNA editing of a group II intron in Oenothera as a prerequisite for splicing; Borner GV et al.; The trans-splicing group II intron c/d in the Oenothera mitochondrial nad1 gene is modified by RNA editing in domain 6 . This C-to-U conversion generates the typical domain 6 structure, which prompted us to speculate that this RNA editing event might be essential for splicing . To test this hypothesis, we investigated the influence of unedited and edited sequences of the Oenothera intron on splicing in vitro . The stem of domain 6 of intron nad1-c/d was transplanted into the autocatalytic yeast intron aI5c, yielding chimeras with the genomic C and the edited U, respectively, 5' of the branchpoint A . When incubated under self-splicing conditions, only the edited chimera was released as a lariat, while the precursor with the genomically coded C remained inactive . Our results support the hypothesis that Oenothera group II intron nad1-c/d cannot be spliced from the primary transcript without previous editing in domain 6.

J Biol Chem, 1995 Mar 17, 270(11), 6141 - 8
A camptothecin-resistant DNA topoisomerase I mutant exhibits altered sensitivities to other DNA topoisomerase poisons; Knab AM et al.; The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex . In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance . To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons . Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells . This correlated with a significant reduction in drug-induced DNA cleavage in vitro . However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II . The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin . In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin . These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.

J Biol Chem, 1995 Mar 17, 270(11), 5849 - 56
Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease; Gimble FS et al.; The PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences . The function of two acidic residues within these motifs, Asp218 and Asp326, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions . All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme . By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity . Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage . These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands . Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position . These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.

J Biol Chem, 1995 Mar 17, 270(11), 5695 - 7
Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system; Fuchs A et al.; The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67phox, p47phox and p40phox as well as a small G protein Rac (for review, see Refs . 1-3) . A truncated form of the p40phox cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47phox as target . This truncated form of p40phox consisting of the Src Homology 3 (SH3) domain to the 3' stop codon was also shown to interact with p67phox in the same system . A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47phox or p67phox . Results show that the SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C terminus of p40phox but not its SH3 domain is involved in the interaction with p67phox.

Nature, 1995 Mar 16, 374(6519), 276 - 80
Replication of transcriptionally active chromatin; Lucchini R et al.; In eukaryotic cells, active genes and their regulatory sequences are organized into open chromatin conformations in which nucleosomes can be modified, disrupted or totally absent . It has been proposed that these characteristic chromatin structures and their associated factors might be directly inherited by the newly synthesized daughter strands during chromosome duplication . Here we show that in the yeast Saccharomyces cerevisiae, replication machinery entering upstream of a transcriptionally active ribosomal RNA gene generates two newly replicated coding regions regularly packaged into nucleosomes, indicating that the active chromatin structure cannot be directly inherited at the replication fork . Whereas the establishment of an exposed chromatin conformation at some newly replicated rRNA gene promoters can occur shortly after the passage of the replication fork, regeneration of the active chromatin structure along the coding region is always a post-replicative process involving disruption of preformed nucleosomes.

Oncogene, 1995 Mar 16, 10(6), 1243 - 7
WT1, the Wilms' tumor suppressor gene product, represses transcription through an interactive nuclear protein; Wang ZY et al.; The Wilms' tumor suppressor gene, wt1, encodes a transcription factor of the zinc finger family . Mutations in WT1 have been detected in subsets of Wilms' tumor and in patients with the Denys-Drash Syndrome . In order to determine how WT1 regulates transcription and perhaps the consequences that mutations in WT1 may have, we established that residues 85-124 and 181-250 of WT1 constitute domains that function independently with a DNA binding domain to repress or activate transcription, respectively, and function equally effectively with heterologous promoters, suggesting the activator and repressor domains interact with nuclear components of general importance . To seek evidence for such components, increasing concentrations of WT1 repressor domain without a zinc finger DNA binding domain were co-transfected with fixed concentrations of wild-type (wt) WT1 and PDGF A-chain promoter/reporter gene constructs . As levels of the repressor domain were increased, a progressive loss of wt WT1 repressor activity and a progressive increase in its activation were observed, suggesting that the repressor domain of WT1 competes with wt WT1 for an interactive protein that is an essential component of the repressor activity of wt WT1 . Because the most common mutation associated with Denys-Drash Syndrome disrupts the zinc finger domains of WT1, the results also suggest that the mutant WT1 may have aberrant DNA binding activity and perhaps function as a dominant negative effector of wt WT1.

Transplantation, 1995 Mar 15, 59(5), 685 - 9
Nitric oxide donors improve gut function after prolonged hypothermic ischemia; Villarreal D et al.; The objective of this study was to determine whether the nitric oxide (NO) donors, spermine NO and 3-morpholinosydonimine-N-ethyl-carbamide (SIN1), alter the mucosal and microvascular responses of the feline small intestine to 6 hr of hypothermic ischemia and 2 hr of normothermic reperfusion . Intestinal mucosal permeability was monitored using the blood-to-lumen clearance of 51Cr-EDTA . Lymph flow and lymphatic protein clearance estimates were used to assess intestinal microvascular fluid filtration and vascular protein leakage, respectively . Spermine NO (0.1 mmol/L) or SIN1 (0.5 mmol/L) was added to the luminal perfusate during the entire reperfusion period . Both NO donors were effective in attenuating the increased mucosal permeability to 51Cr-EDTA and the depressed net water absorption, relative to untreated intestinal preparations exposed to the same protocol . Intestinal lymph flow, lymphatic protein clearance, and capillary hydrostatic pressure were increased by a greater extent in preparations treated with spermine NO . These findings suggest that NO donors may improve mucosal function in intestinal allografts subjected to prolonged hypothermic ischemia . This protective effect on mucosal epithelium appears to be unrelated to an action of the NO donors on the microvasculature.

Cancer Res, 1995 Mar 15, 55(6), 1235 - 8
DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene; Ross GM et al.; The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination . Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant . We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis . These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Eur J Biochem, 1995 Mar 15, 228(3), 878 - 85
Cytochrome c reductase from potato does not comprise three core proteins but contains an additional low-molecular-mass subunit; Jansch L et al.; Analysis of cytochrome c reductase from potato by Tricine/SDS/PAGE reveals 10 bands representing 10 different subunits . In comparison to glycine/SDS/PAGE one additional small protein becomes visible, whereas the three large core proteins are not resolved . The identity of the subunits was determined by immunoblotting and direct sequence determination . Sequence data for the novel small component were used to derive oligonucleotides for probing a potato cDNA-library and isolating corresponding clones . The newly identified subunit is a 6.7-kDa protein, that exhibits significant sequence similarity to a 8.5-kDa subunit of cytochrome c reductase from yeast and the 6.5-kDa iron-sulfur-protein-binding factor from the equivalent enzyme complex from beef . Also the potato 6.7-kDa subunit can be dissociated from the cytochrome c reductase complex together with the iron-sulfur protein . To address the question of whether three or two core subunits occur simultaneously in monomeric cytochrome c reductase complexes from potato, a peptide-specific antibody was generated . The antiserum is capable of discriminating between the 55-kDa and 53-kDa core proteins, which can be separated by glycine/SDS/PAGE and which were previously found to be structurally related . Immunoprecipitations of isolated cytochrome c reductase from potato using this antibody revealed an enzyme complex containing only two core proteins . The simultaneous occurrence of only two core subunits was confirmed by a comparison of the molecular masses of cytochrome c reductase from potato and beef by blue-native-gel electrophoresis . Hence the cytochrome c reductase complexes from potato, beef and yeast have a very conserved subunit composition . The evolutionary implications of these findings are discussed.

Genes Dev, 1995 Mar 15, 9(6), 742 - 55
MEK-2, a Caenorhabditis elegans MAP kinase kinase, functions in Ras-mediated vulval induction and other developmental events; Wu Y et al.; Activated Ras initiates a cascade of sequential phosphorylation events, including the protein kinases Raf, MEK, and MAP kinase . The Let-60 Ras-mediated signal transduction pathway controls vulval induction in Caenorhabditis elegans . Both Lin-45 Raf and Sur-1 MAP kinase have been determined to be essential factors during vulval induction; however, the C . elegans mek gene has not been identified . In this paper, we have cloned a C . elegans mek gene, mek-2, and demonstrated that the MEK-2 protein possesses the biochemical properties of MAP kinase kinases: The C . elegans MEK-2 protein can phosphorylate and activate a human MAP kinase (ERK1), and MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf . The mek-2 gene plays a key role in the let-60 ras-mediated vulval induction pathway, as loss-of-function mutations in the gene (ku114 and h294) significantly reduce the signal transmitted through Ras . mek-2(ku114) completely suppressed the Multivulva (Muv) phenotype of a hyperactive let-60 ras mutation, and animals homozygous for mek-2(ku114) also displayed a partial larval lethal phenotype . Animals homozygous for mek-2(h294) exhibited a highly penetrant sterile and Vulvaless phenotype . Microinjection of a gain-of-function mek-2 mutation resulted in Muv and other mutant phenotypes, whereas microinjection of a dominant-negative mutation not only suppressed the Muv phenotype of an activated let-60 ras mutation but also caused an egg-laying defective phenotype in otherwise wild type animals . Our results demonstrate that mek-2 acts between lin-45 raf and sur-1/mpk-1 in a signal transduction pathway used in the control of vulval differentiation and other developmental events.

EMBO J, 1995 Mar 15, 14(6), 1209 - 20
Transcriptional silencing by the Polycomb protein in Drosophila embryos; Muller J; Polycomb group (Pc-G) proteins act to keep homeotic genes stably and heritably silenced during Drosophila development . Here, it is shown that Polycomb (Pc), one of the Pc-G proteins, acts as a transcriptional silencer in Drosophila embryos if tethered to reporter genes by the DNA binding domain of GAL4 (i.e . as a GAL-Pc fusion protein) . The results suggest that silencing by GAL-Pc requires the C-terminal portion of Pc, but not the chromodomain . If a pulse of Gal-Pc is provided, synthetic reporter genes are repressed, though only transiently . In contrast, reporter genes containing homeotic gene sequences remain stably and heritably silenced in a Pc-G gene-dependent fashion, even when GAL-Pc is no longer present . This implies that GAL-Pc recruits Pc-G proteins to DNA and suggests that maintenance of silencing requires the anchoring of Pc-G proteins to specific cis-regulatory sequences present in homeotic genes . The extent of DNA over which the Pc-G machinery acts is quite selective, as silencing established on one enhancer does not necessarily 'spread' to a juxtaposed synthetic enhancer.

Anal Chem, 1995 Mar 15, 67(6), 1132 - 8
DNA conformational dynamics in polymer solutions above and below the entanglement limit; Shi X et al.; Video microscopy of nucleic acids (DNA) undergoing electrophoresis in hydroxyethyl cellulose (HEC) sieving buffers demonstrates previously unobserved shape-changing interactions between DNA and HEC molecules . We provide the first visual demonstration of entanglement between DNA and one or several discrete HEC molecules, which has been postulated to occur in ultradilute polymer solutions . Typically, nucleic acids appear to become entangled with HEC at a single region only, in both dilute and fully entangled HEC solutions . Fluctuations of the center of mass velocity of a DNA molecule and its correlation with conformation are revealed from analyses of the image data . These observations account for the success of recently reported rapid, high-resolution dc and pulsed-field capillary electrophoretic separations of nucleic acids in ultradilute hydroxyethyl cellulose solutions and hydroxyethyl cellulose/poly(ethylene oxide) solutions.

Biochim Biophys Acta, 1995 Mar 14, 1261(1), 129 - 33
Primary structure of a folate-dependent trifunctional enzyme from Spodoptera frugiperda; Tremblay GB et al.; The dehydrogenase and synthetase activities of the NADP-dependent methylenetetra-hydrofolate dehydrogenase-cyclohydrolase-synthetase are undetectable in extracts of the Spodoptera frugiperda cell line, Sf9 . However, a single cDNA encoding this protein was isolated from a library and sequenced . The deduced amino acid sequence codes for a protein of 933 amino acids in length that shows 59% identity to the human enzyme . The cDNA inserted in the yeast expression vector pVT102-U complements a purine auxotrophic yeast strain lacking this enzyme.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2403 - 7
E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle; Sardet C et al.; The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation . Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130 . In vivo, pRb is found in complex with a subset of E2F components--namely, E2F-1, E2F-2, and E2F-3 . Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system . E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity . However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay . Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable . These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early G1 events including the G0/G1 transition.

Biochemistry, 1995 Mar 14, 34(10), 3268 - 76
Protein thermal denaturation, side-chain models, and evolution: amino acid substitutions at a conserved helix-helix interface; Pielak GJ et al.; Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened . All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries . Mutants with these combinations are biologically functional . Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants . Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation . The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically . Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity . Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions . First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently . Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones . Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3222 - 30
Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences; Taylor WE et al.; Yeast ADR1 contains two Cys2,His2 zinc fingers needed for DNA binding to the upstream activation sequence UAS1, with bases T5T6G7-G8A9G10 in the ADH2 promoter . Potential DNA-contacting amino acid residues at -1, +3, and +6 in the alpha-helical domains of ADR1's fingers one and two include RHR-RLR; however, the latter finger two residues Leu146 and Arg149 had not proved to be crucial for ADR1 binding, even though Leu146-T6 and Arg149-T5 interactions with UAS1 DNA were predicted . We altered Leu146 or Arg149 by PCR cassette mutagenesis, to study ADR1 mutant binding to 16 UAS1 variants of thymine bases T5 and T6 . Mutation of Leu146 to His, making finger two (RLR) like finger one (RHR), decreased binding to wild type UAS1 having T6, but enhanced its binding strength to sequences having purines G6 or A6, similar to binding seen between finger one's His118 and base A9 of UAS1 . Mutating Leu146 to Lys caused this finger two RKR mutant to bind strongly to both G6 and T6, possibly by lysine's amine H-bonding to the carbonyl of guanine or thymine . Specificity of ADR1 for UAS1 with T6 may thus be due to hydrophobic interaction between Leu146 and the T6 methyl group . ADR1 mutants with either His or Lys in the central +3 residue (146) of zinc finger two, which have Arg149 in the +6 alpha-helical position, bind with UAS1 mutant sequences having G5 very strongly, T5 strongly, A5 intermediately, and C5 weakly.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Mar 10, 270(10), 5674 - 9
Biogenesis of ISP6, a small carboxyl-terminal anchored protein of the receptor complex of the mitochondrial outer membrane; Cao W et al.; To study the biogenesis of ISP6, an outer membrane component of the mitochondrial protein translocation complex, two fusion proteins have been made by fusing ISP6 to either the carboxyl- or amino-terminal end of the mouse dihydrofolate reductase (DHFR) . In vitro import experiments showed that when DHFR was placed at the carboxyl-terminal end of ISP6, the resulting fusion protein 6-DHFR inserted into mitochondrial membrane less efficiently than the other form of the fusion proteins . In vivo this fusion protein lost its ability to suppress the temperature-sensitive phenotype of an isp42 mutant, while the other fusion protein DHFR-6, which was found targeted correctly to mitochondria, suppressed the mutant as well as the wild-type ISP6 . Further analysis showed that the binding and insertion of DHFR-6 to mitochondrial outer membrane was not affected by deletion of either of the two mitochondrial protein receptors or by the predigestion of mitochondrial surface proteins prior to import . Additional data indicated that ISP42, which closely associates with ISP6 in the translocation complex, does not likely play the role of a targeting partner for ISP6 . In summary, these data suggest that ISP6 may target to mitochondria by sequences at its carboxyl terminus and that the import process of ISP6 is most likely distinct from that of most other mitochondrial precursors, which are recognized by protein receptors on mitochondrial surface.

J Biol Chem, 1995 Mar 10, 270(10), 5625 - 30
Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor; Kawabata M et al.; The transforming growth factor-beta (TGF-beta) superfamily comprises a number of molecules that are involved in a wide variety of biological processes . Specific receptors for several members of this family have been molecularly identified, forming a new category of transmembrane serine/threonine kinase receptors . The type I and type II receptor interact both physically and functionally, thereby cooperating to generate intracellular signals . The yeast two-hybrid system was used to identify proteins that can interact with the cytoplasmic region of the type I TGF-beta receptor . One of the proteins identified encodes a novel putative serine/threonine kinase receptor . Sequence analysis suggests that this molecule belongs to the type II receptor class . This receptor, however, is distinct from other type II receptors in having an extraordinarily long C-terminal tail region . The pattern of expression in adult tissues is different from that of other known type II receptors; it is highly expressed in heart and liver . In the yeast system, the cytoplasmic regions of different combinations of type I and type II receptors heterodimerize, providing a new cloning strategy for the large number of serine/threonine kinase receptors likely to exist for the many ligands of the TGF-beta superfamily.

J Biol Chem, 1995 Mar 10, 270(10), 5549 - 55
The APC protein and E-cadherin form similar but independent complexes with alpha-catenin, beta-catenin, and plakoglobin; Rubinfeld B et al.; The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin . To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins . beta-Catenin directly binds to APC at high and low affinity sites . alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin . Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin . beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 amino acid repeat sequences . The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor amino-terminal halves of beta-catenin will independently associate with APC, although the amino-terminal half independently binds alpha-catenin . The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins . Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.

J Biol Chem, 1995 Mar 10, 270(10), 5251 - 7
Hormone-dependent transactivation by the human androgen receptor is regulated by a dnaJ protein; Caplan AJ et al.; Genetic studies were performed to examine the role of eukaryotic dnaJ protein, Ydj1p, in the regulated activation of human androgen receptor (hAR) after heterologous expression in Saccharomyces cerevisiae . Hormone-dependent activation of hAR was measured as a function of lacZ reporter gene expression, which was defective in ydj1-151 and ydj1-2 delta null mutant strains compared to the wild type . This defect was not due to receptor misfolding, since hAR in both wild type and mutant strains had a similar capacity to bind hormone . The target for Ydj1p action was determined to be the hAR hormone binding domain since an N-terminal fragment lacking this region was constitutively active in both wild type and ydj1-151 mutant strains . These data correlate hormone dependence of hAR activation with a requirement for Ydj1p function and are consistent with a role for dnaJ proteins in signal transduction by steroid hormone receptors.

Gene, 1995 Mar 10, 154(2), 205 - 9
Isolation and characterization of a cDNA encoding a chicken actin-like protein; Michaille JJ et al.; We report the isolation and characterization of a chicken cDNA which putatively encodes an actin-like protein (chACTL) . This 394-amino-acid (aa) polypeptide shares sequence homology (81, 70 and 67% identical aa, respectively) with three actin-related proteins (ARP) described for Drosophila melanogaster (ARP14D), Caenorhabditis elegans (ACTL) and Saccharomyces cerevisiae (ACT2) . At least six chACTL transcripts were detected in different tissues during chick embryogenesis . Sequence analysis suggests that at least three groups of ARP have been evolutionarily conserved.

Science, 1995 Mar 10, 267(5203), 1488 - 91
Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element; Kirchner J et al.; The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III) . An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target . Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription . This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

J Mol Biol, 1995 Mar 10, 246(5), 576 - 84
Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters; Griffith JD et al.; The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM) . Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples . Excellent agreement among the three methods was obtained . With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed . Non-specific binding to DNA ends was common . The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization . With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end . Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees . Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions.

Nature, 1995 Mar 9, 374(6518), 193 - 6
A kinase-cyclin pair in the RNA polymerase II holoenzyme; Liao SM et al.; The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins . The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD) . The CTD and SRB proteins have been implicated in the response to transcriptional regulators . We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively . Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme . The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo . Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation . Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems . These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

Nature, 1995 Mar 9, 374(6518), 173 - 7
ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion; Hay JC et al.; Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins . ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions . PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and here we report that PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K) . The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming . Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway . The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.

Biochemistry, 1995 Mar 7, 34(9), 3040 - 7
The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: investigation of aliphatic residues; Herrmann L et al.; A series of hydrophilic to hydrophobic surface mutations were prepared at the highly solvent-exposed lysine 73 of iso-1-cytochrome c to assess the ability of such mutants to affect the energetics of the denatured state . In this report, the aliphatic hydrophobics (leucine, isoleucine, valine, alanine, glycine) were studied . The thermodynamic stability of each of these mutants was determined by guanidine hydrochloride denaturation . Both the free energy of unfolding in the absence of denaturant, delta GouH2O, and the slope, m, of a plot of the free energy of unfolding, delta Gou, versus {guanidine hydrochloride} show significant negative correlations with the 1-octanol to water transfer free energy, delta Gtr, of the amino acid side chain at position 73 . A negative correlation with hydrophobicity is consistent with these mutants leading to more extensive hydrophobic clustering in the denatured state, consistent with the predictions of heteropolymer theory for compact denatured states; an effect operating on the native state energetics should produce a positive correlation of delta GouH2O with hydrophobicity . Infrared amide I spectroscopy indicated native state structural perturbations for the glycine 73 and isoleucine 73 mutants . A moderate correlation of delta GouH2O was also found with alpha-helix propensity, suggesting that both hydrophobic effects acting on the denatured state and alpha-helix propensity are affecting the delta GouH2O values for these mutants.

J Mol Biol, 1995 Mar 3, 246(4), 522 - 30
Substrate specificity and assembly of the catalytic center derived from two structures of ligated uridylate kinase; Muller-Dieckmann HJ et al.; Two crystal structures of ligated uridylate kinase from Saccharomyces cerevisiae were determined by X-ray analyses . The ligands were ADP and AMP . Cocrystallization with ATP yielded crystals with ADP at the ATP site and a mixture of AMP and ADP at the NMP site . Cocrystallization with ADP gave rise to a distinct crystal type with ADP at the ATP site, but only AMP at the NMP site . In both cases, the substrates are kept in place by favorable crystal contacts . The structures have been refined to R-factors of 17.8% and 19.6% at resolutions of 2.1 A and 1.9 A, respectively . A comparison with the related cytosolic adenylate kinase from pig disclosed large induced-fit movements on substrate binding and the disassembly of the catalytic center in the absence of substrates . The relatively high side-activity of uridylate kinase for AMP is explained by the finding that the binding pocket is sized for an AMP, but constructed to bind UMP together with a water molecule.

Oncogene, 1995 Mar 2, 10(5), 841 - 7
A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene; Ishioka C et al.; The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae . Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations . In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras . This strain is dependent on the Ha-ras for growth, allowing a simple growth assay for NF1GRD loss-of-function mutations . In a test of our method, two alternatively spliced NF1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutants with amino acid substitutions at highly conserved residues did not . This simple method thus permits the rapid screening for heterozygous germline or somatic NF1GRD mutations . In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymphoblasts from 11 previously untested neurofibromatosis type 1 patients.

Nature, 1995 Mar 2, 374(6517), 91 - 4
Interaction of thyroid-hormone receptor with a conserved transcriptional mediator; Lee JW et al.; The thyroid-hormone receptors are hormone-dependent transcription factors that control expression of many target genes . This regulation is presumably a consequence of hormone-dependent contacts between the receptors and the basal transcription machinery . We used the yeast two-hybrid system to identify a candidate human transcriptional mediator that interacts with both the thyroid-hormone receptor and the retinoid-X receptor in a ligand-dependent fashion . This protein, Trip1 (for thyroid-hormone-receptor interacting protein), shares striking sequence conservation with the yeast transcriptional mediator Sug1 (refs 6, 7) . Here we show that Trip1 can functionally substitute for Sug1 in yeast, and that both proteins interact in vitro with the thyroid-hormone receptor, and with the transcriptional activation domains of yeast GAL4 and of herpes virus VP16.

Biochem J, 1995 Mar 1, 306 ( Pt 2), 367 - 70
Allosteric modulation of oxygen binding to the three human embryonic haemoglobins; Hofmann O et al.; Plasmid based yeast expression systems have been developed for the high-level expression of the three human embryonic haemoglobins Gower I (zeta 2 epsilon 2), Gower II (alpha 2 epsilon 2) and Portland (zeta 2 gamma 2) . Physiochemical characterization of the three product haemoglobins show them to be in the 'native' state . Oxygen-binding studies show that, under what are usually considered physiological conditions, each of the embryonic haemoglobins shows a high oxygen affinity, coupled to a high degree of co-operativity . Allosteric modulation of the oxygen-binding properties of the three haemoglobins in response to organic phosphates and protons has been investigated . The various responses exhibited by the three haemoglobins are rationalized in terms of their amino acid sequences.

Am J Hum Genet, 1995 Mar, 56(3), 640 - 6
Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family; Fridovich-Keil JL et al.; We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system . A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel . Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells . While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism . To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Chem, 1995 Mar, 41(3), 455 - 7
Catecholamine interference with enzymatic determination of nonesterified fatty acids in two commercially available test kits; Carr RE et al.; We present evidence that catecholamines, which are commonly used to stimulate lipolysis in adipose tissue in vitro, interfere with the enzymatic determination of non-esterified fatty acid (NEFA) in two commercially available kits . Measurement of a 100 mumol/L standard with the Wako "NEFA C" test kit was 60% inhibited by 100 mumol/L norepinephrine and was completely inhibited by 100 mumol/L isoproterenol or by 1 mmol/L norepinephrine or epinephrine . Measurement with the Boehringer Mannheim "Free Fatty acids, Half-micro test" was completely inhibited by 100 mumol/L norepinephrine and was also affected by concentrations as low as 0.1 mumol/L . We propose that this effect is due to the catecholamines interfering with a step common to the two kits, the generation of hydrogen peroxide and oxidation of a chromagen; furthermore, this interference appears to be stoichiometric . We also give details of an alternative in-house method, which does not depend on the generation of hydrogen peroxide and is not affected by catecholamines.

J Cell Biol, 1995 Mar, 128(5), 721 - 36
Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication; Powers MA et al.; Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport . Such cell-free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60 . Both p200 and p60 have been found to be components of the nuclear pore . Here, the role of p97 has been investigated . Xenopus p97 was isolated and antisera were raised and affinity purified . Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior . Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116 . An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family . A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family . The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted . The p97-depleted nuclei remained largely competent for nuclear protein import . However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA . ssDNA replication in such extracts remains unaffected . Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects . The possible role(s) of p97 in these nuclear functions is discussed.

Mol Cell Biol, 1995 Mar, 15(3), 1817 - 25
The ligand-binding domains of the thyroid hormone/retinoid receptor gene subfamily function in vivo to mediate heterodimerization, gene silencing, and transactivation; Qi JS et al.; The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions . This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors {RXRs}), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs) . These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs . Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form . The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta . GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA . Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells . However, addition of T3 alone leads to transcriptional activation . These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements . Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors . Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers . This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity . This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements . These studies indicate that the LBD has diverse functional roles in gene regulation.

Mol Cell Biol, 1995 Mar, 15(3), 1536 - 44
An essential domain of the c-myc protein interacts with a nuclear factor that is also required for E1A-mediated transformation; Brough DE et al.; Cell transformation by nuclear oncogenes such as c-myc presumably involves the transcriptional activation of a set of target genes that participate in the control of cell division . The function of a small evolutionarily conserved domain of the c-myc gene encompassing amino acids 129 to 145 was analyzed to explore the relationship between cell transformation and transcriptional activation . Deletion of this domain inactivated the c-myc oncogene for cell transformation while retaining the ability to activate transcription of either myc consensus binding sites or a GAL4-dependent promoter when the c-myc N-terminus was fused to the GAL4 DNA-binding domain . Point mutations that altered a conserved tryptophan (amino acid 136) within this domain had similar effects . Expression of the wt c-Myc N terminus (amino acids 1 to 262) as a GAL4 fusion was a dominant inhibitor of cell transformation by the c-myc oncogene, and this same domain also inhibited transformation by the adenovirus E1A gene . Surprisingly, deletion of amino acids 129 to 145 eliminated the dominant negative activity of GAL4-Myc on both c-myc and E1A transformation . Expression of the GAL4-Myc protein in Cos cells led to the formation of a specific complex between the Myc N terminus and a nuclear factor, and this complex was absent with the dl129-145 mutant . These results suggest that an essential domain of the c-Myc protein interacts with a specific nuclear factor that is also required for E1A transformation.

Mol Cell Biol, 1995 Mar, 15(3), 1324 - 32
Association of the vav proto-oncogene product with poly(rC)-specific RNA-binding proteins; Bustelo XR et al.; We have used the yeast two-hybrid system to isolate proteins that interact with the carboxy-terminal SH3-SH2-SH3 region of Vav . One of the clones encoded heterogeneous nuclear ribonucleoprotein K (hnRNP K), a poly(rC)-specific RNA-binding protein . The interaction between Vav and hnRNP K involves the binding of the most carboxy-terminal SH3 domain of Vav to two proline-rich sequences present in the central region of hnRNP K . Overexpression of Vav in mouse fibroblasts leads to the formation of a stable complex with the endogenous hnRNP K and to the preferential redistribution of this protein to the cytoplasmic fraction . More importantly, Vav and hnRNP K proteins also interact in hematopoietic cells . In addition, Vav associates in vitro with a second 45-kDa poly(rC)-specific RNA-binding protein via its SH3-SH2-SH3 region . These results suggest that Vav plays a role in the regulation of the late steps of RNA biogenesis by modulating the function of poly(rC)-specific ribonucleoproteins.

Mol Cell Biol, 1995 Mar, 15(3), 1318 - 23
A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1; Han L et al.; The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae . The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway . We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference) . The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP . Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras . Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras . Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro . Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized . These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

Mol Cell Biol, 1995 Mar, 15(3), 1265 - 73
A ubiquitin mutant with specific defects in DNA repair and multiubiquitination; Spence J et al.; The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin . To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg . Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates . The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates . The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis . Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme . Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair . However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product . The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair . The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

Mol Cell Biol, 1995 Mar, 15(3), 1220 - 33
The transcriptional activator GCN4 contains multiple activation domains that are critically dependent on hydrophobic amino acids; Drysdale CM et al.; GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae . Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein . We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements . Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4 . One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein . Both domains are partially dependent on the coactivator protein ADA2 . Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4 . At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function . Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16 . The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1 . Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4 . These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.

Mol Cell Biol, 1995 Mar, 15(3), 1203 - 9
ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex; Horiuchi J et al.; Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains . In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together . Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2 . Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N . Silverman, J . Agapite, and L . Guarente, Proc . Natl . Acad . Sci . USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells . ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation . The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo . We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

Hum Mol Genet, 1995 Mar, 4(3), 465 - 9
Evidence from antibody studies that the CAG repeat in the Huntington disease gene is expressed in the protein; Jou YS et al.; The neurodegenerative disorder Huntington disease (HD) appears to be caused by an increase in the number of repeats of the trinucleotide CAG located near the 5' end of the gene . The nucleotide sequences of the cDNA and the gene predict that the HD protein has a molecular weight of 347,000 (3144 amino acids) and that the CAG repeats encode a segment of polyglutamine beginning 17 amino acids from the amino terminus . Because the CAG repeat plays such a critical role in the etiology of the disease, we sought to obtain evidence that the polyglutamine segment is indeed present in the protein . We used two peptides, hd1-peptide (FESLKSFQQ), predicted to lie at amino acid positions 11-19, just amino-terminal to the polyglutamine segment, and hd2-peptide (QQPRNKPLK), predicted to lie at amino acid positions 2531-2539, to induce polyclonal antibodies in NZW rabbits . Both antibodies recognize a protein on Western blots of about 350 kDa in cell lysates from human brain tissue and human and monkey cell lines, including cells from individuals heterozygous and homozygous for the disease . These results suggest that the HD protein in these cells contains the predicted amino terminal segment, and by inference, the segment of polyglutamine, and that the protein is expressed even when only mutant copies of the gene are present . Interestingly, the antibody to hd1-peptide does not recognize the HD protein on Western blots containing lysates from rodent cell lines, whereas the antibody to hd2-peptide does . This discrimination provides a useful means to assay for the presence of the human HD protein in a rodent cell background.

Yeast, 1995 Mar, 11(3), 283 - 92
Sequence, mapping and disruption of CCC2, a gene that cross-complements the Ca(2+)-sensitive phenotype of csg1 mutants and encodes a P-type ATPase belonging to the Cu(2+)-ATPase subfamily; Fu D et al.; We have isolated, sequenced, mapped and disrupted a gene, CCC2, from Saccharomyces cerevisiae . This gene displays non-allelic complementation of the Ca(2+)-sensitive phenotype conferred by the csg1 mutation . Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P-type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease . The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca(2+)-induced death of csg1 mutant cells is related to Cu2+ metabolism . Cells without CCC2 require increased Cu2+ concentrations for growth . Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess Cu2+.

J Antimicrob Chemother, 1995 Mar, 35(3), 373 - 80
Evaluation of a broth microdilution antifungal susceptibility test with a pH indicator: comparison with the broth macrodilution procedures; Fournier C et al.; A broth microdilution antifungal susceptibility test with a pH indicator (bromocresol purple) in a synthetic medium was evaluated . This method measures cellular activity instead of simply a change in biomass . The variations of pH caused by fungal activity were measured by changes in optical densities at 450 nm . The MICs50 obtained were compared with the MICs found by the classical broth macrodilution procedure . In most cases broth micro- and macrodilution MICs were in agreement for amphotericin B, fluconazole, flucytosine and nystatin.

Genomics, 1995 Mar 1, 26(1), 70 - 6
Characterization of the Xiphophorus fish (Teleostei: Poeciliidae) ERCC2/XPD locus; Della Coletta L et al.; We have cloned and sequenced the ERCC2/XPD locus of Xiphophorus maculatus . The human ERCC2/XPD gene is a nucleotide excision repair gene presumed to encode an ATP-dependent DNA helicase . The fish ERCC2/XPD gene is represented on 14.5 kb of genomic DNA and is composed of 23 exons . Within the coding regions, the overall nucleotide identity is 74% compared to the human cDNA . Of 760 amino acids compared between human and fish sequences, 127 differences are observed . Of these differences, 48 residues (38%) represent nonconservative amino acid changes, while 79 (62%) are conservative . The majority (73%) of nonconservative differences between the human and the fish amino acid sequences occur in eight distinct groups comprising only about 10% of the total protein . Overall, the fish and human sequences show 83% amino acid identity and 94% similarity when conservative amino acid substitutions are allowed.

Curr Biol, 1995 Mar 1, 5(3), 306 - 17
Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation; Vogel JL et al.; BACKGROUND: The heat-shock protein Hsp104 plays a crucial role in the survival of cells exposed to high temperatures and other severe stresses, but its specific functions and the biological pathways on which it operates have been unclear . Indeed, very little is known about the specific cellular processes in which any of the heat-shock proteins acts to affect thermotolerance . One essential process that is particularly sensitive to heat in many organisms is the splicing of intervening sequences from mRNA precursors . RESULTS: We have examined the role of Hsp104 in the repair of splicing after disruption by heat shock . When splicing in the budding yeast Saccharomyces cerevisiae was disrupted by a brief heat shock, it recovered much more rapidly in wild-type strains than in strains containing hsp104 mutations . Constitutive expression of Hsp104 promoted the recovery of heat-damaged splicing in the absence of other protein synthesis, but did not protect splicing from the initial disruption, suggesting that Hsp104 functions to repair splicing after heat damage rather than to prevent the initial damage . A modest reduction in the recovery of splicing after heat shock in an hsp70 mutant suggested that Hsp70 may also function in the repair of splicing . The roles of Hsp104 and Hsp70 were confirmed by the ability of the purified proteins to restore splicing in extracts that had been heat-inactivated in vitro . Together, these two proteins were able to restore splicing to a greater degree than could be accomplished by an optimal concentration of either protein alone . CONCLUSIONS: Our findings provide the first demonstration of the roles of heat-shock proteins in a biological process that is known to be particularly sensitive to heat in vivo . The results support previous genetic arguments that the Hsp104 and Hsp70 proteins have different, but related, functions in protecting cells from the toxic effects of high temperatures . Because Hsp104 and Hsp70 are able to function in vitro, after the heat-damaged substrate or substrates have been generated, neither protein is required to bind to its target(s) during heating in order to effect repair.

Glycobiology, 1995 Mar, 5(2), 167 - 73
Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water; Roden L et al.; N-Acetyl-D-{2-3H}glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate . The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine . After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8) . The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen . As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2 . {2-3H}Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to {2-3H}glucosamine 6-phosphate by incubation with hexokinase and ATP . The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.

Biotechniques, 1995 Mar, 18(3), 453 - 7
Improved strategy for large-scale DNA sequencing using DNaseI cleavage for generating random subclones; Demolis N et al.; Large-scale DNA sequencing projects require the use of specific and reliable strategies . Here, we describe an improved strategy using DNaseI cleavage and sequencing strategy using DNaseI cleavage and sequencing from both ends of the plasmid inserts . This strategy yields better results than those obtained using partial digestion with restriction enzymes and cloning in single-stranded vectors.

Genetics, 1995 Mar, 139(3), 1201 - 9
A test of a counting model for chiasma interference; Foss EJ et al.; According to the model of FOSS, LANDE, STAHL and STEINBERG, chiasma interference is a reflection of the requirement for crossovers to be separated by an organism-specific number of potential conversion events without associated crossovers . This model predicts that tetrads with close double crossovers should be enriched for conversion events that themselves are not associated with crossing over . We tested this prediction in Saccharomyces cerevisiae and found it to be unfulfilled.

Plant Mol Biol, 1995 Mar, 27(6), 1109 - 18
Structure and expression of LeMA-1, a tomato protein belonging to the SEC18-PAS1-CDC48-TBP-1 protein family of putative Mg(2+)-dependent ATPases; Prombona A et al.; cDNA clones of a tomato protein, called Lycopersicum esculentum putative Mg(2+)-dependent ATPase (LeMA-1), were isolated from a cDNA library . Sequence comparison of the tomato protein with other genes in the database revealed that the protein is highly homologous to a human protein called TBP-1 and a yeast Tat-binding-analogue protein YTA1A . All three proteins belong to the recently discovered protein family of putative Mg(2+)-dependent ATPases and form within this family a subgroup of proteins involved in controlled protein degradation and possibly also in transcriptional regulation . Expression of the mRNA of LeMA-1 could be monitored in several plant tissues . LeMA-1 is the first member of this subgroup of proteins isolated from plants.

Anal Chem, 1995 Mar 1, 67(5), 784 - 6
Rapid pulsed field capillary electrophoretic separation of megabase nucleic acids; Kim Y et al.; Pulsed field capillary electrophoresis in ultradilute solutions of sieving polymers has been used to separate nucleic acid fragments as long as 1.6 million base pairs . Hydroxyethyl cellulose solutions are used for separations in the size range 8000-50,000 base pairs . Longer chain nucleic acids are separated in mixed hydroxyethyl cellulose/poly(ethylene oxide) solutions . Separations are rapid (12-13 min).

J Magn Reson B, 1995 Mar, 106(3), 253 - 60
13C-coupled relaxation studies of a leucine zipper peptide using polarization-transfer pulse sequences; Brown RA et al.; Three new pulse sequences are described for perturbing magnetization modes in 13C-coupled relaxation experiments via polarization-transfer techniques . Relative to non-polarization-transfer pulse sequences, these new pulse sequences result in carbon NMR spectra with significantly improved signal-to-noise ratios, a condition required for coupled relaxation studies of larger biomolecules . These pulse sequences are used to study molecular tumbling of a 13C-labeled leucine zipper peptide, GCN4-p1, in aqueous solution . Experimental data obtained for the AX2 spin system associated with the 13CH2 moiety of the peptide are fitted to the Favro diffusion model via nonlinear least-squares minimization . The least-squares fits provide values for rotational diffusion coefficients . Diffusion coefficients from relaxation studies performed at different temperatures yield enthalpies for the diffusional motion . Deficiencies in the fits of the relaxation data suggest the need for expanded relaxation models that account for proximate protons in the vicinity of the 13CH2 moiety.

Trends Biochem Sci, 1995 Mar, 20(3), 98 - 101
The protein import receptor of mitochondria; Lithgow T et al.; Protein import into the mitochondria of Saccharomyces cerevisiae depends on two receptor subcomplexes composed of integral outer-membrane proteins . One subcomplex is the MAS37-MAS70 heterodimer, which preferentially recognizes the mature regions of precursor proteins associated with ATP-dependent cytosolic chaperones . The other subcomplex contains the acidic proteins MAS20 and MAS22, which recognize the positively charged targeting sequences of a wide variety of mitochondrial precursors . We propose that the two subcomplexes can act together as a single, multifunctional receptor that binds simultaneously to different regions of a precursor molecule.

J Helminthol, 1995 Mar, 69(1), 13 - 7
Oxidation and reduction of cytochrome c by mitochondrial enzymes of Setaria cervi; Goyal N et al.; A mitochondria-rich fraction isolated from the cuticle-hypodermis-muscle system of Setaria cervi, a bovine filarial parasite, possessed substrate-coupled cytochrome c reductases and cytochrome c oxidase in appreciable activities . All these activities were located predominantly in the membranes . NADH-coupled cytochrome c reductase was more prominent than NADPH- and succinate-coupled reductases . All the three reductases exhibited marked sensitivity to rotenone and antimycin A . Salicylhydroxamic acid strongly inhibited succinate requiring reductase and cytochrome c oxidase, but the other two reductases only mildly . Sodium azide activated the reductases but substantially inhibited the oxidase activity . Potassium cyanide activated the succinate requiring reductase but did not cause any noticeable change in the activities of pyridine nucleotide linked reductases . Anthelmintics also influenced these activities but no definite correlation could be drawn regarding their mode of action.

J Cell Sci, 1995 Mar, 108 ( Pt 3), 927 - 34
A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication; Starborg M et al.; We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle . The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae . We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle . This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle . Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1 . The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication . The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus . Our data support this model and indicate that the murine P1 protein could function as replication licensing factor . The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

J Cell Sci, 1995 Mar, 108 ( Pt 3), 1195 - 204
Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes; Guenette S et al.; To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy suppressors of a conditional mutation in alpha-tubulin . Cells expressing the recessive allele tub1-729 as their sole alpha-tubulin gene grow normally at permissive temperature . However, at 15 degrees C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures . Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type alpha-tubulin genes, TUB1 and TUB3; and BUB3 . BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules . Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729 . The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the 'no microtubule' phenotype . Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not . Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but disruption of the BUB3 gene produces slow growth and benomyl supersensitivity . Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.

Somat Cell Mol Genet, 1995 Mar, 21(2), 91 - 8
Periodicity of eight nucleotides in purine distribution around human genomic CpG dinucleotides; Clay O et al.; Mammalian genomes, unlike the genomes of Drosophila and yeast, are characterized by CpG methylation and concomitant CpG depletion, which is caused by the enhanced mutation rate of 5-methylcytosine . To find out whether local nucleotide sequences around existing methylated CpG dinucleotides have common patterns, we analyzed a large population of CpG-poor regions in human DNA, which are typically methylated . We detected a novel periodic variation in the numbers of purine bases around CpGs in the noncoding parts of these sequences . This periodicity of eight nucleotides gradually diminished over 64 nucleotides on each side of the central CpG . Furthermore, the frequencies of the 5' and 3' nearest neighbors of CpGs in CpG-poor regions were biased towards cytosine and guanine, respectively . Such biased sequence contexts may have helped to stabilize CpGs against depletion during mammalian evolution.

Mol Cell Biol, 1995 Mar, 15(3), 1137 - 43
Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A; Brondyk WH et al.; Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis . Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras) . Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction . These results demonstrate that the interaction is highly specific . However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion . The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase . Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.

J Neurochem, 1995 Mar, 64(3), 1420 - 3
Phosphorylation of tau protein by casein kinase-1 converts it to an abnormal Alzheimer-like state; Singh TJ et al.; The microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease . Both proline-dependent protein kinases (PDPKs) and non-PDPKs are involved in this hyperphosphorylation of tau . Several PDPKs can phosphorylate tau in vitro and induce Alzheimer-like epitopes to many phosphorylation-dependent antibodies . A similar induction has not been reported with non-PDPKs . In this study we have evaluated six non-PDPKs {cyclic AMP-dependent (A-kinase), calcium/phospholipid-dependent (C-kinase), casein kinase-1 (CK-1), casein kinase-2 (CK-2), calcium/calmodulin-dependent protein kinase II, and calcium/calmodulin-dependent protein kinase from rat cerebellum} for their abilities to induce Alzheimer-like epitopes on tau . Such epitopes were induced by A-kinase, C-kinase, CK-1, and CK-2, but the degree of induction achieved by CK-1 was much greater than with the other kinases . These results suggest that CK-1 may play an important role in the conversion of tau from the normal to the abnormal phosphorylation state in Alzheimer's disease.

Protein Eng, 1995 Mar, 8(3), 225 - 36
Neural network system for the evaluation of side-chain packing in protein structures; Milik M et al.; An artificial neural network system is used for pattern recognition in protein side-chain-side-chain contact maps . A back-propagation network was trained on a set of patterns which are popular in side-chain contact maps of protein structures . Several neural network architectures and different training parameters were tested to decide on the best combination for the neural network . The resulting network can distinguish between original (from protein structures) and randomized patterns with an accuracy of 84.5% and a Matthews' coefficient of 0.72 for the testing set . Applications of this system for protein structure evaluation and refinement are also proposed . Examples include structures obtained after the application of molecular dynamics to crystal structures, structures obtained from X-ray crystallography at various stages of refinement, structures obtained from a de novo folding algorithm and deliberately misfolded structures.

Biochemistry, 1995 Feb 28, 34(8), 2694 - 700
Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin; Miller CJ et al.; Yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84 were studied to assess their role in interactions with myosin . In a previous report Dictyostelium actin filaments with residues D24/D25 or E99/E100 replaced with histidines showed complete or partial loss of filament sliding in the in vitro motility assay {Johara, M., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 2127-2131} . In the motility experiments reported here, actin filaments with alanines substituted at D24/D25 or E99/E100 moved in the presence of 0.7% methylcellulose at velocities similar to those of wild-type yeast actin . Without methylcellulose, mutant filaments dissociated from the assay surface upon addition of ATP with little or no sliding detected . In contrast to this, filaments with alanines substituted at D80/D81 or E83/K84 were motile in the presence and absence of methylcellulose . Direct binding measurements involving cosedimentation of D24A/D25A and E99A/E100A actins with myosin subfragment-1 (S-1) in the presence of ATP revealed 3- and 2-fold decreases in their binding constants, respectively, compared to wild-type actin . In the absence of ATP all yeast actins had a similar affinity for S-1 . A large decrease in the activation of S-1 ATPase was observed for both D24A/D25A and E99A/E100A actins . The D80A/D81A and E83A/K84A actin filaments showed normal S-1 binding and activation of ATPase activity . These results demonstrate the involvement of the D24/D25 and E99/E100 charged residues in the weak binding of myosin to actin and reveal that D80/D81 and E83/K84 residues in the 79-92 helix do not modulate actomyosin interactions.

Gene, 1995 Feb 27, 154(1), 115 - 8
Isolation and characterization of the arginase-encoding gene (arg) from Coccidioides immitis; Pan S et al.; The arginase (ARG)-encoding gene (arg) of Coccidioides immitis, a human fungal pathogen, was cloned and sequenced . Both the genomic and cDNA sequences are provided . The transcription start point and poly(A) sites were confirmed . The arg gene, which was located on chromosome II of C . immitis by Southern hybridization, is a single-copy gene with two introns and a 966-bp ORF which translates a 322-aa protein of 35.1 kDa . The deduced ARG protein showed 44% identity and 68% similarity to the Saccharomyces cerevisiae ARG.

Biochem Biophys Res Commun, 1995 Feb 27, 207(3), 910 - 5
Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol; Kelly SL et al.; Azole antifungal compounds inhibit sterol 14 alpha-demethylase . They are used extensively for the treatment of immunocompromised patients where fungal infection is common and often results in death . Resistance to the compounds is emerging, particularly in fungal pathogens obtained from AIDS patients undergoing prolonged therapy . We show here that cell growth arrest correlates with the accumulation of 14 alpha-methyl-ergosta-8,24(28)-dien-3 beta,6 alpha-diol in a yeast strain with a sterol 14 alpha-demethylase gene disruption, which mimics stringent treatment conditions . Cells can overcome the effect of such a block by a suppressor mutation in sterol delta 5,6 desaturation and acquire azole resistance . Plasmid-based complementation of sterol 14 alpha-demethylase defect does not alter the azole susceptibility of strains containing these suppressor mutations, showing resistance is due entirely to the delta 5.6 desaturase defect.

Cell, 1995 Feb 24, 80(4), 533 - 41
Multiple Ras functions can contribute to mammalian cell transformation; White MA et al.; We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors . These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components . The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction . These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

Science, 1995 Feb 24, 267(5201), 1183 - 5
Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line; Lees-Miller SP et al.; The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK . Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins . The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK . These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.

Mol Gen Genet, 1995 Feb 20, 246(4), 445 - 54
Mutations in the mitochondrial split gene COXI are preferentially located in exons: a mapping study of 170 mutants; Netter P et al.; We have analysed the precise location of a large number (170) of mutations affecting the structural gene for subunit I of the cytochrome c oxidase complex . This gene, COXI, is 12.9 kb long and the major part of the sequence (i.e . 11.3 kb) is composed of introns . Several conclusions can be drawn from this study: (1) A significant proportion (84/170) of the mutations cannot be assigned to a single position within the gene by deletion mapping, in spite of clearly being located in it . These mutations are probably large deletions or multiple mutations . (2) Four mutants carry distant double mutations, which have been individually localized . (3) Eighty-two mutants have lesions that are restricted to very short regions of the gene and we therefore conclude that they are most probably due to single hits; amongst these single mutations, 41 are unambiguously located in exons and 28 in introns . This result implies that, at least in this particular split gene, the probability of selection of a mutant phenotype in an exon is, on the average, 13.3 times greater than in an intron, in spite of the existence, within most of these introns, of open reading frames specifying intronic proteins . The evolutionary significance and biological implications of these results are discussed.

J Biol Chem, 1995 Feb 17, 270(7), 3154 - 9
Biochemical and genetic definition of the cellular protease required for HIV-1 gp160 processing; Franzusoff A et al.; The surface glycoproteins of enveloped viruses bind to target cell receptors and trigger membrane fusion for infection . The human immunodeficiency virus 1 (HIV-1) envelope glycoprotein gp120 (CD4 binding protein) and gp41 (transmembrane fusion protein) are initially synthesized as a gp160 precursor . The intracellular cleavage of gp160 by a host cell protease during transit through the secretory pathway is essential for viral activities such as infectivity, membrane fusion, and T-cell syncytium formation . We report that gp160 biogenesis, protein processing, and cell-surface expression have been successfully reproduced in the yeast Saccharomyces cerevisiae . Genetic and biochemical approaches are used for defining that the unique cellular protease, Kex2p, is directly responsible for HIV-gp160 processing in yeast, in vivo and in vitro . The yeast system described in this report represents a powerful strategy for identifying, characterizing and inhibiting the host T-cell protease essential for HIV infectivity and AIDS.

J Chromatogr A, 1995 Feb 17, 693(1), 162 - 6
Simultaneous structure-activity determination of disulfiram photolysis products by on-line continuous-flow liquid secondary ion mass spectrometry and enzyme inhibition assay; Benson LM et al.; Disulfiram (DSF) is used in the treatment of recovering alcoholics and exerts its effect by inhibiting the enzyme aldehyde dehydrogenase (ALDH) . We analyzed a mixture of products derived photochemically from DSF with on-line microbore HPLC-continuous-flow liquid secondary ion mass spectrometry (HPLC-CF-LSI-MS) . By utilizing the post-HPLC column split of solvent flow, a small proportion (ca . 5%) was sent directly into the mass spectrometer, and the remainder was collected . Simultaneous MS analysis and enzyme inhibition studies on ALDH were then possible . Furthermore, using HPLC-CF-LSI-MS-MS, we were able to structurally characterize an interesting sulfine compound that inhibited ALDH.

Genes Dev, 1995 Feb 15, 9(4), 481 - 90
RNA polymerase II subunit RPB9 is required for accurate start site selection; Hull MW et al.; The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12 . Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown . We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection . Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo . Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9 . In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro . A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro . RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.

Genes Dev, 1995 Feb 15, 9(4), 437 - 54
Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen; He F et al.; Rapid turnover of nonsense-containing mRNAs in yeast in dependent on the product of the UPF1 gene (Upf1p) . Mutations in UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs . To identify other integral components of this decay pathway, we have employed a two-hybrid screen, seeking those cellular factors that specifically interact with Upf1p . Screening of yeast genomic libraries identified six genes encoding potential Upf1p-interacting proteins . These include four previously uncharacterized genes, NMD1-4 (nonsense-mediated mRNA decay), DBP2, a gene encoding a putative RNA helicase with homology to mammalian p68 RNA helicase, and SNP1, a gene encoding a U1 snRNP 70-kD protein homolog . In this paper we report the identification and characterization of NMD2, a yeast gene that encodes a specific Upf1p-interacting protein . Disruption of NMD2 yields a nonsense-mediated mRNA decay phenotype identical to that obtained in UPF1-deletion strains, indicating that the NMD2 gene product (Nmd2p) is a new factor in the nonsense-mediated mRNA decay pathway . Deletion analysis demonstrated that the acidic carboxyl terminus of Nmd2p constituted the Upf1p-interacting domain . High-level expression of a fragment of Nmd2p containing this domain had a dominant-negative effect on nonsense-mediated mRNA decay when the protein was localized the cytoplasm but not when it was localized to the nucleus, indicating that this decay pathway has a cytoplasmic component . The association of a dominant-negative phenotype with a gene fragment identified in a two-hybrid screen suggests a generalized approach to confirming the function of genes identified in such screens.

Eur J Biochem, 1995 Feb 15, 228(1), 138 - 43
Isolation of the catalytic subunit of a membrane-bound H(+)-pyrophosphatase from pea stem mitochondria; Zancani M et al.; The catalytic subunit of a membrane-bound pyrophosphatase was purified by electroendosmotic preparative electrophoresis from etiolated pea stem mitochondria . The enzyme was identified as a single peak relatively pure, because only a very limited number of polypeptides were detectable by SDS/PAGE of the active fractions . The pyrophosphatase was associated to a band with a molecular mass of 35 kDa, showing a specific activity of 0.7 mumol Pi . mg-1 protein . min-1 (37 degrees C, pH 8.0) and an apparent Km value of 200 microM . The hydrolytic activity required Mg2+, was inhibited by imidodiphosphate (HNO6P2Na4), Ca2+, F- and was stimulated by phospholipids . Cardiolipin, phophatidylcholine and phosphatidylethanolamine had the maximal activating effect . The isolated protein is very similar to the catalytic subunit of pyrophosphatases isolated from rat liver (beta-subunit) and Saccharomyces cerevisiae mitochondria.

EMBO J, 1995 Feb 15, 14(4), 820 - 32
Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs; Jandrositz A et al.; A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs . An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6 . It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP . By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24 . Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24 . Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes . Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site . Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.

EMBO J, 1995 Feb 15, 14(4), 791 - 800
Purification of the sequence-specific transcription factor CTCBF, involved in the control of human collagen IV genes: subunits with homology to Ku antigen; Genersch E et al.; The common promoter region of the human collagen type IV genes COL4A1 and COL4A2 comprises a C5TC7 sequence ('CTC box') which is specifically recognized by the recently identified transcription factor CTC box binding factor (CTCBF) involved in the control of divergent transcription of the two genes . This factor has now been purified by affinity chromatography on heparin-agarose and CTC-Sepharose . The CTCBF contains two subunits, CTC75 and CTC85, with molecular weights of 75 and 85 kDa, respectively . Sequence analysis of LysC-derived peptides of the two subunits revealed identity or close homology to p70 and p80 subunits of the human autoantigen Ku . The sequence-specific binding CTCBF represents a presumably tetrameric complex composed of two CTC75/85 heterodimers with an apparent molecular weight of 360-400 kDa . UV crosslinking experiments, the use of Ku-specific antibodies in gel retardation assays and immunoblotting proved that both subunits are involved in sequence-specific interaction with the CTC box motif . The tetrameric complex dissociates in a concentration-dependent manner to CTC75/85 heterodimers which now bind sequence independently to DNA . Three lines of evidence indicate that TATA binding protein (TBP) is additionally involved in the formation of CTCBF: (i) TBP can be detected in purified CTCBF; (ii) the addition of recombinant TBP stimulates formation of the CTCBF-DNA complex; and (iii) antibodies directed against TBP interfere strongly with the formation of the specific protein-DNA complex . The results presented support the idea that the subunits CTC75 and CTC85 (identical or homologous to p70 and p80 of the Ku antigen) are integral parts of CTCBF, and give a first indication of the importance of TBP in the formation of CTCBF.

Biochem J, 1995 Feb 15, 306 ( Pt 1), 29 - 33
Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209; Iwasaki M et al.; The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates . P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209 . Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite . On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced . Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209 . Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem . Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis . These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 949 - 53
A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase; Krynetski EY et al.; Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine:thiopurine S-methyltransferase, EC 2.1.1.67) activity exhibits genetic polymorphism, with approximately 0.33% of Caucasians and African-Americans inheriting TPMT deficiency as an autosomal recessive trait . To determine the molecular genetic basis for this polymorphism, we cloned the TPMT cDNA from a TPMT-deficient patient who had developed severe hematopoietic toxicity during mercaptopurine therapy . Northern blot analysis of RNA isolated from leukocytes of the deficient patient demonstrated the presence of TPMT mRNAs of comparable size to that in subjects with high TPMT activity . Sequencing of the mutant TPMT cDNA revealed a single point mutation (G238-->C), leading to an amino acid substitution at codon 80 (Ala80-->Pro) . When assessed in a yeast heterologous expression system, this mutation led to a 100-fold reduction in TPMT catalytic activity relative to the wild-type cDNA, despite a comparable level of mRNA expression . A mutation-specific PCR amplification method was developed and used to detect the G238-->C mutation in genomic DNA of the propositus and her mother . This inactivating mutation in the human TPMT gene provides insights into the genetic basis for this inherited polymorphism in drug metabolism.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1187 - 91
Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes; Pemberton LF et al.; We have characterized a protein with an estimated molecular mass of 130 kDa that is contained in a highly enriched yeast nuclear pore complex (NPC) fraction . Partial amino acid sequence from this protein has led us to a previously identified open reading frame on chromosome XI of Saccharomyces cerevisiae encoding a protein of 133 kDa . Due to its coenrichment with NPCs during cell fractionation and the phenotype observed in the disrupted strain, we propose to term the gene encoding this protein NUP133 . Cells carrying a disrupted copy of NUP133 were temperature sensitive for growth . In addition, abnormal clustering of NPCs was observed . This phenotype is similar to that previously observed in the disruption of another nucleoporin gene, NUP145 . We speculate that the gene product of NUP133, Nup133p, may functionally overlap with the NUP145 gene product, Nup145p, and that these proteins may be involved in maintaining the position of the NPC within the nuclear envelope.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1182 - 6
G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro; Deshaies RJ et al.; In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division . To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract . A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase . Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase . This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1003 - 7
Hepatitis B virus transactivator protein X interacts with the TATA-binding protein; Qadri I et al.; Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP) . These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex . We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro . While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect . Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX . The minimal region in pX sufficient for its interaction with TBP includes aa 110-143 . Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX . The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP . These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters . This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.

Biochim Biophys Acta, 1995 Feb 14, 1228(1), 95 - 8
The nuclear-encoded MSS2 gene is involved in the expression of the mitochondrial cytochrome-c oxidase subunit 2 (Cox2); Simon M et al.; Saccharomyces cerevisiae cells carrying the mss2-1 pet mutation contain no Cox2 protein (cytochrome-c oxidase subunit 2), through COX2 transcripts are synthesized and processed normally . Gene MSS2 was cloned and sequenced . It is localized on chromosome IV . The Mss2 protein does not show any significant homology with published sequences.

Biochemistry, 1995 Feb 14, 34(6), 1948 - 58
Enzymic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase; Wang Y et al.; We have obtained several cysteine mutants in or around the cytochrome c peroxidase binding domain of rat and yeast iso-1 cytochrome c by site-directed mutagenesis . These cysteine residues were specifically labeled with the bifunctional photoactive cross-linker 4-azidophenacyl bromide (APB) . 1:1 covalent complexes of cytochrome c peroxidase and cytochrome c were generated by cross-linking these specifically labeled cytochromes c to cytochrome c peroxidase, and the 1:1 complexes were purified . Steady-state kinetic studies of the purified 1:1 complexes with free yeast and horse cytochromes c showed the following: (1) Cytochrome c peroxidase has two distinct catalytic sites--a high-affinity and a low-affinity site . (2) Other than the difference in affinity, the binding of substrate at the low-affinity site is similar to that at the high-affinity site, with yeast cytochrome c interacting more strongly than the horse protein, the binding of both substrates being sensitive to ionic strength, and both sites able to transfer electrons . (3) HPLC chromatography of purified 1:1 complex showed multiple forms of 1:1 complexes, supporting the idea of multiple possible interactions between cytochrome c and the high-affinity site on cytochrome c peroxidase . (4) An allosteric or electrostatic effect exists between the two substrate binding sites, the binding of cytochrome c to the high-affinity site decreasing the binding affinity of the low-affinity site to cytochrome c . The higher the equilibrium binding affinity of the mutant cytochrome c to the peroxidase, the larger the apparent allosteric/electrostatic effect when that mutant protein is covalently bound to the high-affinity site of the enzyme . Furthermore, different locations of the covalently bound cytochrome c at the high-affinity site on the enzyme surface result in different degrees of allosteric/electrostatic effect . The presence of two active sites on the enzyme allows a simple interpretation of some of the differences in the steady-state kinetic behavior of cytochrome c peroxidase with horse and yeast iso-1 cytochrome c.

FEBS Lett, 1995 Feb 13, 359(2-3), 179 - 83
Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against alpha-oxidation of phytanic acid in rat liver mitochondria; Singh H et al.; The two branched chain fatty acids pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) were converted to co-enzyme A thioesters by rat liver mitochondrial outer membranes . However, these branched chain fatty acids could not be converted to pristanoyl and phytanoyl carnitines, respectively, by mitochondrial outer membranes . As expected, the unbranched long chain fatty acids, stearic acid and palmitic acid, were rapidly converted to stearoyl and palmitoyl carnitines, respectively, by mitochondrial outer membranes . These observations indicate that the branched chain fatty acids could not be transported into mitochondria . The data presented strongly suggest that in rat liver, alpha-oxidation of phytanic acid occurs in organelles other than mitochondria.

FEBS Lett, 1995 Feb 13, 359(2-3), 159 - 63
Nucleotide binding by the synapse associated protein SAP90; Kistner U et al.; The rat synapse associated protein SAP90 is a member of a superfamily of potential guanylate kinases localized at cell-cell contact sites . This superfamily includes the synapse associated protein SAP97, a close relative of SAP90, the Drosophila tumor suppressor gene product dlg-Ap, the mammalian zonula occludens proteins ZO-1 and ZO-2 and the erythrocyte protein p55 . Here we show that SAP90 specifically binds GMP in the micromolar range while binding to ATP, GDP and ADP is at a much lower affinity (10-25 mM), whether or not binding is detected for other guanine and adenine nucleotides . No guanylate kinase activity of SAP90 was detected under our experimental conditions . The importance of the GMP binding capacity per se and an evolutionary role for conserving of the guanylate kinase domain in this superfamily are discussed.

FEBS Lett, 1995 Feb 13, 359(2-3), 129 - 32
Cooperation of the molecular chaperone Ydj1 with specific Hsp70 homologs to suppress protein aggregation; Cyr DM; Ydj1p, a cytosolic DnaJ homolog from Saccharomyces cerevisiae, is demonstrated to function as a molecular chaperone . Purified Ydj1p formed complexes with non-native polypeptides and suppressed protein aggregation . Ydj1p cooperated with Ssa Hsp70 proteins in the prevention of protein aggregation, but not with the Ssb Hsp70 proteins . Cooperation between these different molecular chaperones was only observed in the presence of hydrolyzable ATP and correlated with the ability of Ydj1p to stimulate the ATPase activity of the Hsp70 homolog with which it was paired . The regulatory and chaperone activities of a eukarytic DnaJ homolog thus act together to assist Hsp70 in modulating the conformation of proteins.

Nucleic Acids Res, 1995 Feb 11, 23(3), 507 - 14
Insertion site specificity of the transposon Tn3; Davies CJ et al.; The Tn3-deletion method {Davies and Hutchison, Nucleic Acids Res . 19, 5731-5738, (1991)} was used to sequence a 9.4 kb DNA fragment . Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer . Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified . There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (approximately 37% of sites are potential targets) . In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently . The 935 bp cold spot showed no unusual features when analysed with the consensus sequence . The consensus can be used to accurately predict likely insertion sites in a new sequence . Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised . These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot . We conclude that some other factor, such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.

J Biol Chem, 1995 Feb 10, 270(6), 2630 - 5
Site-directed mutagenesis of vacuolar H(+)-pyrophosphatase . Necessity of Cys634 for inhibition by maleimides but not catalysis; Kim EJ et al.; A characteristic feature of the vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) of plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents . Previous investigations in this laboratory have demonstrated that the primary site for substrate-protectable covalent modification of the V-PPase by 14C-labeled NEM maps to a single M(r) 14,000 V8 protease fragment (V8(14)K) (Zhen, R.-G., Kim, E . J., and Rea, P . A . (1994) J . Biol . Chem . 269, 23342-23350) . Here, we describe site-directed mutagenesis of the cDNA encoding the V-PPase from Arabidopsis thaliana, its heterologous expression in Saccharomyces cerevisiae and single substitution of all 9 conserved Cys residues to either Ser or Ala . In all cases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM . By contrast, and in complete agreement with the results of peptide mapping experiments, substitution of Cys634, the sole conserved cysteine residue encompassed by V8(14)K, with Ser or Ala generates enzyme that is insensitive to NEM but active in both PPi hydrolysis and PPi-dependent H+ translocation . The specific requirement for Cys634 for inhibition by NEM and the dispensability of all of the conserved Cys residues, including Cys634, for V-PPase function indicate that the inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys634 rather than direct participation of this amino acid residue in catalysis.

J Biol Chem, 1995 Feb 10, 270(6), 2601 - 6
Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR; McMillan NA et al.; The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties . Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166 . We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding . Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60 . Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type) . We have also identified a number of other residues that influence dsRNA binding to varying degrees . Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo . However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.

FEBS Lett, 1995 Feb 6, 359(1), 93 - 6
Mitochondrial presequences can induce aggregation of unfolded proteins; Endo T et al.; We have studied the interactions between various synthetic peptides and two model unfolded proteins, reduced alpha-lactalbumin and reduced and carboxymethylated alpha-lactalbumin . We found that mitochondrial presequences could induce aggregation of the unfolded alpha-lactalbumins but not of the native alpha-lactalbumin . The presequence-induced aggregation of unfolded alpha-lactalbumin was dependent on electrostatic interactions and on the amphiphilicity of the presequences . Since positive charge and amphiphilicity are necessary for the targeting function of mitochondrial presequences, presequence-induced aggregation may be responsible for the instability of mitochondrial precursor proteins and may need to be inhibited by binding factors in the cytosol.

FEBS Lett, 1995 Feb 6, 359(1), 27 - 30
Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)-tris-bipyridyl derivative of cytochrome c as electron donor; Zaslavsky DL et al.; Yeast iso-1-cytochrome c covalently modified at cysteine-102 with (4-bromomethyl-4'-methylbipyridine){bis(bipyridine)}Ru2+ (Ru-102-Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles . Rapid kinetics of membrane potential generation by the enzyme following flash-induced photoreduction of Ru-102-Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipyridyl)3 (RuBpy) as the photoreductant {D.L . Zaslavsky et al . (1993) FEBS Lett . 336, 389-393} . At low ionic strength, when Ru-102-Cyt c forms a tight electrostatic complex with COX, flash-activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles . The initial rapid phase is virtually identical to the 50 microsecond transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA . CuA reduction by Ru-102-Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme . A millisecond phase (tau ca . 4 ms) following the 50 microsecond initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru-102-Cyt c as electron donors; however, this phase is small in the absence of added H2O2 . In addition to these two transients, the flash-induced electrogenic response in the presence of Ru-102-Cyt c reveals a large slow phase of delta psi generation not observed with RuBpy . This phase is completely quenched upon inclusion of 100 microM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru-102-Cyt c2+ generated by the flash in a solution.

Gene, 1995 Feb 3, 153(1), 135 - 9
A phosphate-repressible, high-affinity phosphate permease is encoded by the pho-5+ gene of Neurospora crassa; Versaw WK; The pho-5+ gene of Neurospora crassa, which encodes a high-affinity phosphate permease, has been cloned and analyzed . The deduced ORF of 1707 nucleotides is interrupted by a single 63-nt intron and codes for a protein of 569 amino acids (aa) . This aa sequence has 48% identity with the high-affinity phosphate transporter of Saccharomyces cerevisiae, PHO84 . The pho-5 null mutants have no obvious phenotype . Strains which contain a null mutation in pho-4, which encodes an additional high-affinity phosphate permease {Bowman et al., J . Bacteriol . 153 (1983) 292-296}, also have no obvious phenotype . However, strains containing mutations in both pho-5 and pho-4 are unable to grow under phosphate-restrictive conditions.

Mol Immunol, 1995 Feb, 32(3), 213 - 27
Molecular cloning of major and minor allergens of Alternaria alternata and Cladosporium herbarum; Achatz G et al.; The two moulds, Alternaria alternata and Cladosporium herbarum, are recognized as major causes of fungal allergies . Cloning, sequencing and heterologous expression of the allergens of the two moulds is a necessary step in understanding fungal allergy and in the development of new and improved methods of diagnosis and therapy . The seven new mould allergens presented here represent four new allergen proteins: aldehyde dehydrogenase (ALDH), enolase, YCP4 (previously found as a Saccharomyces cerevisiae protein of unknown function), and the acidic ribosomal protein, P2 . Three of them (ALDH, YCP4 and P2) were found to be allergens in both fungi, Alternaria and Cladosporium . All allergens found so far are cytoplasmic proteins and are rather well conserved in evolution even when comparing distant species . Most of the allergens have "household" functions (ALDH, enolase) . One allergen (P2) is a homolog of a very highly conserved human lupus erythematodes (LE) antigen . None of the fungal allergens is clearly related to other known non-fungal allergens.

Immunity, 1995 Feb, 2(2), 137 - 47
Effect of TAP on the generation and intracellular trafficking of peptide-receptive major histocompatibility complex class I molecules; Day PM et al.; Using a fluorescein-conjugated antigenic peptide, peptide-receptive H-2Kb MHC class I molecules were found throughout the secretory pathways of RMA cells and peptide transporter (TAP)-deficient derivative cells (RMA/S) . RMA/S cells displayed higher levels of intracellular peptide-receptive molecules, while, surprisingly, RMA cells expressed 3- to 5-fold more cell surface peptide-receptive molecules . Metabolic radiolabeling of Kb-associated oligosaccharides with {1-3H}galactose demonstrated that despite a large difference in the fraction of Kb molecules in native conformation in detergent extracts, Kb transport rates from the trans-Golgi complex to the surfaces of RMA and RMA/S cells were similar . Thus, although considerable numbers of class I alpha chains reach the RMA/S cell surface, they are a less productive source of peptide-receptive molecules than class I molecules synthesized by TAP-expressing RMA cells, suggesting paradoxically that TAP functions to increase the amount of peptide-receptive molecules at the cell surface.

Eur J Biochem, 1995 Feb 1, 227(3), 707 - 14
Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing . The importance of the first residue C-terminal of the cleavage site; Rholam M et al.; The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined . Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites . These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded . Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated . But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage . The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues . A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite . We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.

J Cell Biol, 1995 Feb, 128(4), 485 - 98
Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein required for nuclear positioning and completion of asexual development; Fischer R et al.; Many fungi are capable of growing by polarized cellular extension to form hyphae or by isotropic expansion to form buds . Aspergillus nidulans anucleate primary sterigmata (apsA) mutants are defective in nuclear distribution in both hyphae and in specialized, multicellular reproductive structures, called conidiophores . apsA mutations have a negligible effect on hyphal growth, unlike another class of nuclear distribution (nud) mutants . By contrast, they almost completely block entry of nuclei into primary buds, or sterigmata (bud nucleation), produced during development of conidiophores . Failure of the primary sterigmata to become nucleated results in developmental arrest and a failure to activate the transcriptional program associated with downstream developmental steps . However, occasionally in mutants a nucleus enters a primary bud and this event relieves the developmental blockage . Thus, there is a stringent developmental requirement for apsA function, but only at the stage of primary bud formation . apsA encodes a 183-kD coiled-coil protein with similarity to Saccharomyces cerevisiae NUM1p, required for nuclear migration in the budding process.

J Cell Biol, 1995 Feb, 128(4), 455 - 66
Zip1-induced changes in synaptonemal complex structure and polycomplex assembly; Sym M et al.; The yeast Zip1 protein is a component of the synaptonemal complex (SC), which is an elaborate macromolecular structure found along the lengths of chromosomes during meiosis . Mutations that increase the length of the predicted coiled coil region of the Zip1 protein show that Zip1 influences the width of the SC . Overexpression of the ZIP1 gene results in the formation of two distinct types of higher order structures that are found in the nucleus, but not associated with chromatin . One of these structures resembles the polycomplexes that have been observed in many organisms and are thought to be aggregates of SC components . The second type of structure, which we have termed "networks," does not resemble any previously identified SC-related structure . Assembly of both polycomplexes and networks can occur independently of the Hop1 or Red1 protein, which are thought to be SC components . Our results demonstrate that Zip1 is a structural component of the central region of the SC . More specifically, we speculate that Zip1 is a component of the transverse filaments that lie perpendicular to the long axis of the complex.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 981 - 6
A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor; Kristensen C et al.; 1 . To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid . The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I . The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors . The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin . Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I . 2 . The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand . This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site {Kjeldsen, Andersen, Wiberg, Rasmussen, Schaffer, Balschmidt, Moller and Moller (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 4404-4408} . 3 . In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.

Mol Cell Biol, 1995 Feb, 15(2), 809 - 23
Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover; Hagan KW et al.; Several lines of evidence indicate that the processes of mRNA turnover and translation are intimately linked and that understanding this relationship is critical to elucidating the mechanism of mRNA decay . One clear example of this relationship is the observation that nonsense mutations can accelerate the decay of mRNAs in a process that we term nonsense-mediated mRNA decay . The experiments described here demonstrate that in the yeast Saccharomyces cerevisiae premature translational termination within the initial two-thirds of the PGK1 coding region accelerates decay of that transcript regardless of which of the stop codons is used . Nonsense mutations within the last quarter of the coding region have no effect on PGK1 mRNA decay . The sequences required for nonsense-mediated mRNA decay include a termination codon and specific sequences 3' to the nonsense mutation . Translation of two-thirds of the PGK1 coding region inactivates the nonsense-mediated mRNA decay pathway . This observation explains why carboxyl-terminal nonsense mutations are resistant to accelerated decay . Characterization of the decay of nonsense-containing HIS4 transcripts yielded results mirroring those described above, suggesting that the sequence requirements described for the PGK1 transcript are likely to be a general characteristic of this decay pathway . In addition, an analysis of the decay intermediates of nonsense-containing mRNAs indicates that nonsense-mediated mRNA decay flows through a pathway similar to that described for a class of wild-type transcripts . The initial cleavage event occurs near the 5' terminus of the nonsense-containing transcript and is followed by 5'-->3' exonucleolytic digestion . A model for nonsense-mediated mRNA decay based on these results is discussed.

Mol Cell Biol, 1995 Feb, 15(2), 731 - 41
p34Cdc28-mediated control of Cln3 cyclin degradation; Yaglom J et al.; Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division . The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation . Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3) . The C-terminal tail of Cln3 functions as a transferable signal for degradation . Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover . The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature . A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein . These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

Chem Senses, 1995 Feb, 20(1), 61 - 8
The taste-active regions of monellin, a potently sweet protein; Somoza JR et al.; Monellin, a protein found in the berries of the West African plant Dioscoreophyllum cumminsii, is one of the most potently sweet compounds known . The native three-dimensional structure of monellin is required for sweetness, and this protein has been the subject of intense research in an attempt at understanding the structural basis for its taste activity . We have used structure-based site-directed mutagenesis to delineate the taste-active site(s) of monellin, and we present these results, along with similar work from M . Kohmura, Y . Ariyoshi and coworkers, in the light of the three-dimensional structure of this protein . The mutagenesis work suggests that at least four residues, located N-terminal to the alpha-helix, form part of a taste-active region of monellin . In addition, there is evidence that a second region, formed by residues in the fourth and fifth beta-strands, may also be contributing to monellin's activity.

Int J Pept Protein Res, 1995 Feb, 45(2), 106 - 15
Synthesis of alpha-factor analogues containing photoactivatable and labeling groups; Jiang Y et al.; Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing both p-benzoyl phenylalanine (Bpa), a photoactivatable group, and 3-(mono- or di-iodo-4-hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid-phase methodologies on a {phenylacetamido}-methyl (PAM) resin . Bpa was introduced into the peptides using Bpa-hydroxybenzotriazole active ester during peptide chain assembly . Biotinylated alpha-factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage . Iodinated HPP was incorporated by acylating free peptides with Bolton-Hunter reagent (3-{diiodo-4-hydroxyphenyl}propanoic acid hydroxysuccinimide ester) in N,N-dimethylformamide and borate buffer (pH 8.0) solutions . Purification of all peptides to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase mu-Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase . All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry . Two analogues, alpha-(diiodotyrosine)-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr-O H, and epsilon-(diiodo-HPP)-Lys-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr -OH, were one twentieth to one-fortieth as active as a alpha-factor, and exhibited approximately one order of magnitude lower affinity to the alpha-factor receptor . The results suggest that these two analogues are alpha-factor agonists and that they can be used as probes of the alpha-factor receptor.

Proteins, 1995 Feb, 21(2), 161 - 4
Crystallization and preliminary X-ray diffraction studies of an a1/alpha 2/DNA ternary complex; Li T et al.; Crystals have been obtained of a ternary complex containing the yeast a1/alpha 2 homeodomain heterodimer bound to a 21-base pair DNA site containing two 5' overhanging bases at each end . The crystals are grown from cobaltic hexamine and form in space group P6(1) or P6(5) with a = b = 133 A, c = 45.4 A . Crystals that are flash-frozen at -179 degrees C diffract to 2.7 A along the c-axis and to 2.4 A in perpendicular directions . The crystals contain one protein-DNA complex in the crystallographic asymmetric unit.

Eur J Cell Biol, 1995 Feb, 66(2), 127 - 35
Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly; Satoh M et al.; The Ku antigen (p70/p80 heterodimer) is the DNA binding component of a DNA-dependent serine/threonine kinase (DNA-PK), the catalytic activity of which is carried by a 350 kDa polypeptide (p350) . In the present studies, the assembly of p70, p80, and p350 was investigated in human K562 (erythroleukemia) cells, and rabbit (RK13) or murine (L-929) cells infected with recombinant vaccinia viruses directing the synthesis of human p70 and p80 . Pulse-chase analysis and density gradient centrifugation revealed a pool of free p70 subunits in K562 cells that dimerized within minutes with newly synthesized p80, whereas Ku became associated with newly synthesized p350 1 to 4 h after the onset of p70/p80 heterodimer assembly . A stable pool of free p80 subunits was not detected, and newly synthesized p80 was degraded rapidly (t1/2 < 1.5 h) unless it became incorporated into a p70/p80 dimer . The explanation for the absence of unassembled p80 subunits in K562 cells was investigated further by expressing human p70 and p80 individually or together in Ku-deficient RK13 or L-929 cells infected with recombinant vaccinia viruses p70-vacc and/or p80-vacc . As in uninfected K562 cells, the t1/2 of the free recombinant human p80 subunit expressed in RK13 cells was < 1.5 h unless it was "rescued" by dimerization with p70 . The t1/2 of human p70 as well as p70/p80 heterodimers was > 16 h in RK13 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipids, 1995 Feb, 30(2), 163 - 7
Dietary oligofructose lowers triglycerides, phospholipids and cholesterol in serum and very low density lipoproteins of rats; Fiordaliso M et al.; The present study was aimed at answering the question why feeding rats an oligofructose (OFS) supplemented diet could cause a significant reduction in plasma lipid levels . Daily administration of a 10% (w/w) OFS-containing diet to normolipidemic male rats resulted in a decrease in plasma triglycerides, phospholipids and cholesterol . The triglyceride-lowering effect was observed after one week and lasted for at least 16 wk and was associated with a reduction in plasma very low density lipoproteins, indicating that the hypolipidemic effect of OFS may be due to changes in liver lipid metabolism . We therefore tested whether OFS feeding modified the capacity of the liver to synthesize triglycerides from free fatty acids . Hepatocytes isolated from livers of control and OFS-fed rats were incubated in the presence of {1-14C}palmitate, and both intracellular and extracellular {14C}triglyceride formation were quantified . We found that chronic feeding of an OFS-supplemented diet to rats significantly reduced the capacity of isolated hepatocytes to synthesize triglycerides from palmitate . The results suggest that, like other soluble dietary fibers, OFS significantly alters liver lipid metabolism, resulting over time in a significant reduction in plasma triglyceride, phospholipid and cholesterol levels.

Int J Biochem Cell Biol, 1995 Feb, 27(2), 215 - 24
Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography; Weber B et al.; Cyclic AMP affinity chromatography applied to various mammalian tissue extracts yielded two proteins in addition to the regulatory subunits of protein kinase . This paper characterizes these proteins and provides a simple procedure for their preparation . The polypeptides (36 kDa and a 19 kDa/21 kDa doublet) were isolated from the cAMP matrix by sequential elution with cAMP solutions of increasing concentrations . Microsequencing was accomplished following chemical or enzymic degradation of isolated polypeptides . Partial amino acid sequences of the 36 kDa protein and analyses of its enzymic activity indicated identity with glyceraldehyde-3-phosphate dehydrogenase whilst the lower MW protein proved to be identical with mammalian nucleoside diphosphate kinase subunits . In both cases, binding to cAMP appeared to occur at the nucleotide (NAD and ATP, respectively) sites . In conclusion, we present a one step-procedure, applicable to tissue and cell extracts, which allows the simultaneous isolation of both glyceraldehyde-3-phosphate dehydrogenase and nucleoside diphosphate kinase . This procedure may help to elucidate the multiple functions of these two important enzymes.

Hum Mol Genet, 1995 Feb, 4(2), 157 - 61
Characterization of a mutation that abolishes quinone reduction by electron transfer flavoprotein-ubiquinone oxidoreductase; Beard SE et al.; Two mutant alleles of the gene encoding electron transfer flavoprotein-ubiquinone oxidoreductase were identified and characterized in fibroblasts from a patient with glutaric acidemia type II . One of these alleles is a C-T transition in the donor site of an intron that causes skipping of a 222 bp exon . Included in the missing 74 amino acids is C561, which is predicted to be one of the four cysteine ligands of the 4Fe4S cluster . This mutant allele does not encode a stable ETF-QO in human fibroblasts but, when expressed in Saccharomyces cerevisiae, the mutant ETF-QO is relatively stable and properly targeted to and processed by mitochondria . The mutant protein lacks ubiquinone reductase activity, but does accept electrons from ETF in the catalyzed disproportionation of ETF semiquinone . These data suggest that in the normal protein the flavin center accepts electrons from ETF and that the 4Fe4S cluster reduces ubiquinone . Deleting the 74 amino acids also alters the association between the protein and membrane such that the mutant ETF-QO cannot be extracted from the membrane using the same conditions used for wild type ETF-QO . A site directed mutant that contains only the single amino acid substitution, C561A, exhibits the same catalytic behavior as the deletion mutant, supporting the hypothesis regarding the specific functions of the two redox centers . It is, however, solubilized by the same conditions as wild type ETF-QO.

Curr Opin Genet Dev, 1995 Feb, 5(1), 24 - 30
Rho-related proteins: actin cytoskeleton and cell cycle; Ridley AJ; The past year has produced an abundance of data on the function and regulation of Rho-related GTP-binding proteins . In mammalian cells, it has been shown that Rho is required for contractile ring assembly at cell division, as well as for regulating extracellular factor induced actin reorganization . In addition, many new regulators and/or potential targets for Rho, Rac and Cdc42 have been characterized, including several oncogene products, protein kinases and signal transducing proteins in mammalian cells, and genes defined by cell cycle or bud emergence mutations in yeast . These provide further connections between Rho-related proteins, signal transduction pathways and changes in actin organization during cell cycle entry and progression.

Curr Genet, 1995 Feb, 27(3), 195 - 200
Reversions to respiratory competence of omnipotent sup45 suppressor mutants may be caused by secondary sup45 mutations; Mironova LN et al.; The molecular nature of the sup45 respiratory deficient omnipotent suppressor, and of three reversions to respiratory competence which removed the suppressor effect of the initial mutation, was examined . All reversions were caused by secondary sup45 mutations which indicates a direct connection between sup45 "respiratory" and "translational" functions . Computer analysis showed the local changes of Sup45 protein characteristics in the suppressor strain and revertants in comparison to the wild-type protein . The distribution of mutant sites in relation to evolutionary conserved, and tentatively functional, regions in the Sup45 protein is discussed.

Nat Genet, 1995 Feb, 9(2), 115 - 25
Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders; Dodt G et al.; The peroxisome biogenesis disorders (PBDs) are lethal recessive diseases caused by defects in peroxisome assembly . We have isolated PXR1, a human homologue of the yeast P . pastoris PAS8 (peroxisome assembly) gene . PXR1, like PAS8, encodes a receptor for proteins with the type-1 peroxisomal targeting signal (PTS1) . Mutations in PXR1 define complementation group 2 of PBDs and expression of PXR1 rescues the PTS1 import defect of fibroblasts from these patients . Based on the observation that PXR1 exists both in the cytosol and in association with peroxisomes, we propose that PXR1 protein recognizes PTS1-containing proteins in the cytosol and directs them to the peroxisome.

Toxicol Lett, 1995 Feb, 76(1), 63 - 9
Mechanism of phthalate-induced inhibition of hepatic mitochondrial beta-oxidation; Winberg LD et al.; Studies show that peroxisome proliferators inhibit mitochondrial beta-oxidation of fatty acids . However, mechanism(s) of this inhibitory effect has not been identified . This study was undertaken to delineate such mechanism(s) . Ketogenesis was significantly diminished in perfused livers from rats pre-treated with diethylhexyl phthalate (DEHP) compared with livers from control rats . Monethylhexyl phthalate (MEHP; 200 microM), a primary metabolite of DEHP and a known peroxisome proliferator, inhibited the oxidation of palmitic acid as well as its acyl-CoA and acylcarnitine derivatives in isolated mitochondria by about 50-60% . Similar concentrations of MEHP also inhibited mitochondrial respiration of succinate and malate plus glutamate . However, respiration of ascorbate was not influenced by MEHP . Considering the assembly of the mitochondrial respiratory chain, these data indicate that phthalates inhibit fatty acid metabolism as a result of inhibiting the respiratory chain at the level of the cytochrome c reductase . This effect may represent an early step in the mechanism by which phthalates cause hepatic peroxisome proliferation.

J Biochem (Tokyo), 1995 Feb, 117(2), 414 - 9
Oligomeric structures required for complement activation of serum mannan-binding proteins; Yokota Y et al.; Serum mannan-binding protein (S-MBP) comprises a series of homooligomers, and activates complement when it binds to appropriate carbohydrate ligands . In this study, the structural requirements necessary for complement activation were examined for rat, rabbit, and human S-MBPs . On SDS-PAGE under non-reducing conditions, the S-MBPs gave three major bands: large, middle, and small oligomers . Since three subunits (23-25 kDa) form a triple helix (the base structural unit) at the collagen-like domain within the S-MBP molecule, it was estimated that human and rabbit S-MBPs comprise a mixture of pentamers, tetramers, and trimers of the respective structural units . In contrast, rat S-MBP is composed of tetramers, trimers, and dimers . The large and middle oligomers were almost equal in their ability to activate complement, whereas the small oligomers had very low or no activity . Upon digestion with bacterial collagenase, the large and small oligomers were degraded almost completely . In contrast, the middle oligomers remained largely intact, and the surviving middle oligomers retained complete ability to activate complement . The degraded product, trimers of the carbohydrate recognition domain (CRD), did not show any complement activating activity . These data indicate that not only the structural integrity of the S-MBP collagen-like domain and CRD, but also a unique conformational structure present in the middle oligomers are critically important for carbohydrate-mediated complement activation.

Braz J Med Biol Res, 1995 Feb, 28(2), 169 - 81
Trehalose metabolism--new horizons in technological applications; Panek AD; Trehalose is responsible for the survival of anhydrobiotic organisms when under stress . Trehalose is a unique, non-reducing, extremely stable disaccharide which is able to protect proteins and membranes from damage caused by freezing, high temperatures and dehydration . Yeasts accumulate large amounts of trehalose and constitute excellent models for studying the response of eucaryotic cells to diverse stresses . The regulation of trehalose metabolism is reviewed and new technological applications for preservation of biological materials are discussed.

Biochem Pharmacol, 1995 Jan 31, 49(3), 305 - 13
Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells; Benchokroun Y et al.; Aurintricarboxylic acid (ATA) is a polyanionic, polyaromatic compound which has been shown to inhibit apoptotic cell death in various cell types induced by a variety of factors . Since ATA is known to be a general inhibitor of nuclease activities in vitro (ID50S ranging from 2 to 50 microM), the in vivo effects are usually attributed to inhibition of endogenous endonuclease activities . We show herein that ATA is a potent inhibitor of the nuclear enzyme DNA topoisomerase II . ATA inhibits the catalytic activity of purified yeast topoisomerase II with an ID50 of approx . 75nM as measured by relaxation assays . ATA does not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex") as do other inhibitors of this enzyme such as 4'-(9-acridinylamino)-methane sulfon-m-anisidide (amsacrime), 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-gluco pyr anoside) (etoposide) and ellipticines . In contrast, cleavable complex formation induced by amsacrine and etoposide is trongly inhibited in the presence of ATA . ATA also prevents the binding of topoisomerase II to DNA and inhibits topoisomerase II-catalysed ATP hydrolysis . The ability of ATA to interfere with more than one step in t he catalytic cycle of DNA topoisomerase II may explain its unusual potency as an inhibitor of this enzyme . ATA reduces the number of amsacrine-induced DNA-protein complexes in intact DC-3F Chinese hamster fibrosarcoma cells and protects these cells from the cytotoxic action of amsacrine . The effects of ATA on DNA-protein complex formation in living cells appear to be due to the direct interaction of the drug with topoisomerase II, since similar results are found when nuclei from untreated DC-3F cells are exposed to amsacrine after a short preincubation with ATA . Cells resistant to 9-hydroxyellipticine, which have been shown to possess altered topoisomerase II activity, are approx . 5-fold more resistant to ATA than the sensitive parental cells as shown by colony formation essays . We conclude that ATA is a potent inhibitor of topoisomerase II and that the drug interacts with topoisomerase II in living cells . Our findings raise the possibility that the protective effects of ATA towards apoptotic cell death might, at least in part, involve DNA topoisomerase II.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 890 - 4
Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen; Boubnov NV et al.; Two ionizing radiation-sensitive (IRs) and DNA double-strand break (DSB) mutants, sxi-3 and sxi-2, were shown to be severely deficient in a DNA end binding activity, similar to a previously described activity of the Ku autoantigen, correlating with the xrs (XRCC5) mutations . Cell fusions with xrs-6, another IRs, DSB repair-deficient cell line, defined these sxi mutants in the XRCC5 group . sxi-3 cells have low expression levels of the p86Ku mRNA . Introduction of the Ku p86 gene, but not the p70 Ku gene, complemented the IRs, DNA end binding, and variable (diversity) joining {V(D)J} recombination signal and coding junction deficiencies of sxi-3 . Thus, the p86 Ku gene product is essential for DSB repair and V(D)J recombination.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 739 - 43
Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction; Johnson FB et al.; The Drosophila homeotic selector (HOM) genes encode a family of DNA binding transcription factors that specify developmental fates of different body segments by differentially regulating the activity of downstream target genes . A central question is how the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro . Specificity could be achieved by differential interactions with protein cofactors . The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax, abdominal-A, and perhaps other HOM genes . By using a yeast two-hybrid system, we demonstrate selective interaction of the extradenticle homeodomain protein with certain Ultrabithorax and abdominal-A proteins but not with an Antennapedia protein or a more distant homeodomain protein . Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts . The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms, and this variable region appears to affect the interaction detected in the assay.

Cell, 1995 Jan 27, 80(2), 285 - 91
Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death; Yang E et al.; To extend the mammalian cell death pathway, we screened for further Bcl-2 interacting proteins . Both yeast two-hybrid screening and lambda expression cloning identified a novel interacting protein, Bad, whose homology to Bcl-2 is limited to the BH1 and BH2 domains . Bad selectively dimerized with Bcl-xL as well as Bcl-2, but not with Bax, Bcl-xs, Mcl-1, A1, or itself . Bad binds more strongly to Bcl-xL than Bcl-2 in mammalian cells, and it reversed the death repressor activity of Bcl-xL, but not that of Bcl-2 . When Bad dimerized with Bcl-xL, Bax was displaced and apoptosis was restored . When approximately half of Bax was heterodimerized, death was inhibited . The susceptibility of a cell to a death signal is determined by these competing dimerizations in which levels of Bad influence the effectiveness of Bcl-2 versus Bcl-xL in repressing death.

Nature, 1995 Jan 26, 373(6512), 360 - 2
A B-cell coactivator of octamer-binding transcription factors; Gstaiger M et al.; The octamer motif (ATGCAAAT) paradoxically plays a central role in mediating the activity of both B-cell specific and ubiquitous promoters . It has been widely assumed that the predominantly lymphoid-restricted octamer-binding factor Oct-2 mediates tissue-specific promoter activity, whereas the ubiquitously expressed Oct-1 mediates general promoter activity, but this view has been challenged . Here we use a modified yeast one-hybrid assay to isolate a B-cell factor, Bob1, which associates with either Oct-2 or Oct-1 . In transfection experiments, this factor boosts Oct-1-mediated promoter activity and to a lesser extent, that of Oct-2 . This coactivation is strictly dependent on the specific interaction with Oct-1 or Oct-2 because deletion of the octamer motif abolishes coactivation . We conclude that Bob1 could represent a new tissue-specific transcriptional coactivator which may convert a ubiquitously expressed transcription factor to a cell-type-specific activator.

Mol Cell Biochem, 1995 Jan 26, 142(2), 163 - 70
Fatty acid delta 5 desaturation in rat liver cell nuclei; Ves-Losada A et al.; Activity of one of the key enzymes involved in arachidonic acid (20:4 n-6) biosynthesis, the delta 5 desaturase, was found in rat liver cell nuclei . Up to now, it has been shown that the fatty acid desaturases are located exclusively in the endoplasmic reticulum . Similarly to what happens with microsomal enzyme the nuclear delta 5 desaturase enzyme was only fully active in the presence of a cytosolic factor . In this condition it reached a specific activity of 50 pmol 20:4 n-6 formed/min/mg of protein . This fact would imply that purified nuclei like purified microsomes lack a soluble cytosol factor necessary for the total desaturation reaction expression . Besides the nuclear delta 5 desaturase has an optimal pH of 7.6 and is inhibited by 1 or 10 mM KCN . Low long chain acyl-CoA synthetase activity that catalyzes the formation of 20:3 n-6-CoA, was also found in liver nuclei . This step would be essential in nuclear desaturation since when ATP and/or CoA (necessary for the acylation reaction) are omitted from the incubation mixture, the desaturation reaction does not take place.

FEBS Lett, 1995 Jan 23, 358(2), 153 - 7
The rsp5-domain is shared by proteins of diverse functions; Hofmann K et al.; A novel, unusually small, and highly conserved domain of modular intracellular proteins is described . The domain was first recognized as three repeats in the yeast rsp5 gene product and named thereafter . The rsp5 protein is thought to interact with nuclear proteins but also contains a C2 domain typical for cytoplasmic proteins . Further analyses revealed several additional occurrences of this domain in diverse protein classes, including cytoplasmic signal transduction proteins, gene products interacting with the transcription machinery, structural proteins like dystrophin, and a putative RNA helicase.

J Biol Chem, 1995 Jan 20, 270(3), 1449 - 54
Stimulation of the DNA-dependent protein kinase by RNA polymerase II transcriptional activator proteins; Peterson SR et al.; The DNA-dependent protein kinase (DNA-PK) phosphorylates RNA polymerase II and a number of transcription factors . We now show that the activity of DNA-PK is directly stimulated by certain transcriptional activator proteins, including the human heat shock transcription factor 1 (HSF1) and a transcriptionally active N-terminal 147 amino acid GAL4 derivative . Stimulation of DNA-PK activity required specific sequences in the activator proteins outside the minimal DNA binding domains . The stimulation of DNA-PK activity also required DNA and was greater with DNA containing relevant activator binding sites . Comparison of different HSF binding fragments showed that optimal stimulation occurred when two HSF binding sites were present . Stimulation with HSF and GAL4 was synergistic with Ku protein, another regulator of DNA-PK activity . DNA-PK is tightly associated with the transcriptional template, and an increase in its activity could potentially influence transcription through the phosphorylation of proteins associated with the transcription complex.

Oncogene, 1995 Jan 19, 10(2), 381 - 7
The retinoblastoma susceptibility gene product regulates Myc-mediated transcription; Adnane J et al.; The protein product of the retinoblastoma tumor suppressor gene (RB) has been demonstrated to bind c-Myc protein (Myc) in vitro . To determine whether RB regulates Myc transcriptional activity in vivo, GAL4-Myc chimeric expression plasmids were generated and cotransfected with a RB expression plasmid and a GAL4-dependent reporter plasmid . RB stimulated GAL4-Myc-mediated transcription, dependent upon a domain(s) in the amino-terminus of Myc . The stimulation of Myc-mediated transcription by RB was cell-type specific and was inhibited by SV40 T-antigen, but not by a T-antigen mutant defective in RB-binding . Moreover, RB mutants containing mutations in domain B of RB pocket were significantly reduced in their ability to stimulate GAL4-Myc mediated transcription . To determine whether RB and Myc interact in vivo either directly or indirectly, a two hybrid system was used where GAL4-Rb and Myc-VP16 expression constructs were cotransfected with a GAL4-dependent reporter plasmid . A significant increase of GAL4-dependent transcription was observed, dependent upon the presence of both GAL4-Rb and Myc-VP16 fusion proteins . Mutational analysis of the Myc-VP16 chimeric proteins suggests that the amino-terminus of Myc is essential for the interaction with RB . These results demonstrate that RB can regulate Myc-mediated transcription in vivo in a cell-type specific manner through protein-protein interactions.

Proc Natl Acad Sci U S A, 1995 Jan 17, 92(2), 527 - 31
Transcytosis-associated protein (TAP)/p115 is a general fusion factor required for binding of vesicles to acceptor membranes; Barroso M et al.; Transcytosis-associated protein (TAP) is found on transytotic vesicles (TCVs) and is required for their fusion with the target membrane . We developed a cell-free assay capable of differentiating targeting/binding of TCVs to membrane from later fusion events . We found that TAP mediates stable association of TCVs with the target membrane . The sequence of rat liver TAP (959-amino acid open reading frame) encodes a protein that contains (i) an N-terminal region (amino acids 1-649), (ii) an internal region with several coiled-coil stretches (amino acids 650-930), and (iii) a C-terminal acidic region (amino acids 931-959) . Comparisons between TAP and other sequences indicate that TAP is identical to p115, a protein involved in cis to medial Golgi transport, and homologous to Uso1p, a yeast protein involved in endoplasmic reticulum to Golgi transport . Our findings suggest that TAP/p115/Usop1 is a general factor acting within the secretory and endocytic pathways to bind transport vesicles prior to membrane fusion.

Biochem Biophys Res Commun, 1995 Jan 17, 206(2), 724 - 30
Glutathione reductase and lipoamide dehydrogenase have opposite stereospecificities for alpha-lipoic acid enantiomers; Pick U et al.; The reduction of exogenous alpha-lipoic acid to dihydrolipoate by mammalian cells and tissues confers additional antioxidant protection to the cell . Both (R+) and (S-) isomers of alpha-lipoic acid were analyzed as substrates with glutathione reductase from several sources and with mammalian lipoamide dehydrogenase . Mammalian glutathione reductase catalyzed faster reduction of (S)-lipoic acid (1.4-2.4-fold greater activity) than of (R)-lipoic acid, whereas lipoamide dehydrogenase had a very marked preference for (R)-lipoic acid (18-fold greater activity) over (S)-lipoic acid . Mammalian glutathione reductase showed better affinity for (S)-lipoic acid substrate; Km values were 3.5 mM for (S)-lipoic acid and and 7 mM for (R)-lipoic acid . Glutathione reductase from yeast reduced lipoic acid less efficiently than the mammalian enymes, had a Km for both stereoisomers of about 10 mM, and showed little stereospecificity . Although (S)-lipoic acid is not formed in nature, these findings indicate that exogenous (S)-lipoic acid may have a useful role as an antioxidant for mammalian systems.

EMBO J, 1995 Jan 16, 14(2), 303 - 12
Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway; Deshaies RJ et al.; Recombinant G1 cyclin Cln2p can bind to and stimulate the protein kinase activity of p34CDC28 (Cdc28p) in an extract derived from cyclin-depleted and G1-arrested Saccharomyces cerevisiae cells . Upon activating Cdc28p, Cln2p is extensively phosphorylated and conjugated with multiubiquitin chains . Ubiquitination of Cln2p in vitro requires the Cdc34p ubiquitin-conjugating enzyme, Cdc28p, protein phosphorylation and unidentified factors in yeast extract . Ubiquitination of Cln2p by Cdc34p contributes to the instability of Cln2p in vivo, as the rate of Cln2p degradation is reduced in cdc34ts cells . These results provide a molecular framework for G1 cyclin instability and suggest that a multicomponent, regulated pathway specifies the selective ubiquitination of G1 cyclins.

Biochem J, 1995 Jan 15, 305 ( Pt 2), 591 - 7
Effects of tetradecylthiopropionic acid and tetradecylthioacrylic acid on rat liver lipid metabolism; Skrede S et al.; Studies of effects of 4-thia-substituted fatty acid analogues on rat liver lipid metabolism are described . With isolated hepatocytes tetradecylthiopropionate was shown to divert {1-14C}oleate from beta-oxidation into esterification, the total amount of {1-14C}oleate metabolized remaining unchanged . Tetradecylthiopropionyl-CoA was a good substrate for mitochondrial carnitine palmitoyltransferases I and II (EC 2.3.1.21), acyl-CoA oxidase (EC 1.3.3.6), for the microsomal (but not mitochondrial) glycerophosphate acyltransferase (EC 2.3.1.15), and for long-chain acyl-CoA dehydrogenase (EC 1.3.99.3) . In isolated hepatocytes, its 4-thia-trans-2-enoic derivative, tetradecylthioacrylate, inhibits both beta-oxidation of, and incorporation of, {1-14C}oleate into lipids . In rat liver mitochondria tetradecylthiocrylate inhibited beta-oxidation . The degree of inhibition was not markedly increased by preincubation with tetradecylthioacrylate . Tetradecylthioacrylyl-CoA was a poor substrate for carnitine palmitoyltransferase I, and inhibited carnitine palmitoyltransferase II, microsomal glycerophosphate acyltransferase and acyl-CoA oxidase . It is concluded that the inhibitory effects of tetradecylthiopropionyl-CoA are expressed intramitochondrially, whereas primary sites of inhibition by tetradecylthioacrylyl-CoA are extramitochondrial.

J Immunol, 1995 Jan 15, 154(2), 961 - 71
Analysis of the autoantibody response to fibrillarin in human disease and murine models of autoimmunity; Takeuchi K et al.; Fibrillarin, a component of the U3 RNP particle, is a target for the spontaneously arising autoantibodies in human scleroderma and a monoclonal autoantibody (72B9) derived from the autoimmune mouse strain (NZB x NZW) F1 . Autoantibodies against fibrillarin can also be induced in H-2s mice by treatment with mercuric chloride (HgCl2) . The objective of this study was to compare the spontaneously occurring anti-fibrillarin autoantibody response with the autoantibody response induced by HgCl2 treatment . Immunofluorescence microscopy on human HEp2, mouse 3T3, and Xenopus XIK-2 cells, immunoblotting with use of nuclear extract from human MOLT-4, mouse 3T3, and Xenopus XIK-2 cells, and immunoprecipitation with use of in vitro translation products of RNA transcripts from yeast fibrillarin cDNA were used in this analysis . Both spontaneous and induced autoantibodies displayed common reactivity in that, irrespective of the antigenic source, they gave the same nucleolar immunofluorescence pattern and a restricted immunoblotting reactivity targeting predominantly the 34-kDa protein fibrillarin . Immunoprecipitation of N- and C-terminal truncated fibrillarin constructs also demonstrated a common pattern of reactivity . All Abs precipitated a fragment comprising amino acids 1-312 but not a smaller fragment containing amino acids 1-257 . The majority of sera could not precipitate an N-terminal truncated molecule consisting of amino acids 157-327 . These immunoprecipitation experiments support recognition of a common epitope requiring both N- and C-regions, which may be exemplified by the reactivity of murine monoclonal anti-fibrillarin autoantibody 72B9 . Our results indicate that spontaneous human and toxin-induced murine autoantibodies to fibrillarin share common reactivity against this highly conserved nucleolar protein.

J Biol Chem, 1995 Jan 13, 270(2), 949 - 57
DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract; Singhal RK et al.; The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway . We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro . A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil . A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction . ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required . Next, the base excision repair system was reconstituted using partially purified components . Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement . We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not . These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

J Biol Chem, 1995 Jan 13, 270(2), 815 - 22
Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells; Sabers CJ et al.; The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase . Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase . These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast . In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts . In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts . Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines . The latter results suggest that mTOR is a relevant target of rapamycin in these cells . Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex . The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively . These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

Cell, 1995 Jan 13, 80(1), 29 - 39
DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint; Navas TA et al.; Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint . We have isolated a mutant, dun2, defective for both of these responses . DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon) . Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage . Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases . We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.

Cell, 1995 Jan 13, 80(1), 21 - 8
Different forms of TFIIH for transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome; Svejstrup JQ et al.; Yeast TFIIH that is active in transcription can be dissociated into three components: a 5-subunit core, the SSL2 gene product, and a complex of 47 kDa, 45 kDa, and 33 kDa polypeptides that possesses protein kinase activity directed towards the C-terminal repeat domain of RNA polymerase II . These three components can reconstitute fully functional TFIIH, and all three are required for transcription in vitro . By contrast, TFIIH that is highly active in nucleotide excision repair (NER) lacks the kinase complex and instead contains the products of all other genes known to be required for NER in yeast: RAD1, RAD2, RAD4, RAD10, and RAD14 . This repairosome is not active in reconstituted transcription in vitro and is significantly more active than any of the constituent polypeptides in correcting defective repair in extracts from strains mutated in NER genes.

Science, 1995 Jan 13, 267(5195), 232 - 4
A salt-sensitive 3'(2'),5'-bisphosphate nucleotidase involved in sulfate activation; Murguia JR et al.; Overexpression of a yeast gene, HAL2, allows the cells to tolerate higher than normal extracellular salt concentrations . HAL2 encodes a 3'(2')5'-bisphosphate nucleotidase that serves to remove the end products of sulfate transfer during cellular metabolism . The enzyme is inhibited by lithium and sodium and is activated by potassium . Metabolic systems that are sensitive to salt, as well as those governing osmolyte synthesis and ion transport, offer routes by which genetic engineering can be used to improve the tolerance of various organisms to salt.

Virology, 1995 Jan 10, 206(1), 773 - 6
The cDNA sequence of Trichomonas vaginalis virus-T1 double-stranded RNA; Tai JH et al.; Trichomonas vaginalis virus (TVV) is a nonsegmented double-stranded (ds) RNA virus that infects the pathogenic protozoan T . vaginalis . To study the virus, we cloned the genomic ds RNA of a TVV-T1 isolate and obtained a contiguous 4647-bp cDNA sequence . Two overlapping genes separated by a + 1 reading frame shift were identified on the plus strand but none on the complementary strand RNA in this sequence . The upstream gene probably encodes a 75-kDa capsid protein, and the downstream gene encodes an 86-kDa polypeptide which is probably the viral RNA-dependent RNA polymerase (RDRP) . A potential ribosomal slippage heptamer (C CUU UUU) was identified within the 14-nt overlap of the two genes . The genomic organization and RDRP sequence in TVV-T1 exhibit similarity to those of Saccharomyces cerevisiae virus and Leishmania RNA virus, suggesting that these viruses originate from common ancestry, but are only distantly related to Giardia lamblia virus.

Virology, 1995 Jan 10, 206(1), 116 - 25
NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein; O'Neill RE et al.; We used the yeast interactive trap system to identify a cellular protein which interacts with the nucleoprotein of influenza A viruses . This protein, nucleoprotein interactor 1 (NPI-1) is the human homolog of the yeast protein SRP1 . SRP1 was previously identified as a suppressor of temperature-sensitive RNA polymerase I mutations (R . Yano, M . Oakes, M . Yamaghishi, J . Dodd, and M . Nomura, Mol . Cell . Biol . 12, 5640-5651, 1992) . A full-length cDNA clone of NPI-1 was generated from HeLa cell poly A + RNA . The viral nucleoprotein, which had been partially purified from influenza A/PR/8/34 virus-infected embryonated eggs, could be coprecipitated from solution by glutathione agarose beads complexed with a bacterially expressed glutathione-S-transferase-NPI-1 fusion protein, confirming the results of the yeast genetic system . Antisera raised against NPI-1 identified a 60-kDa polypeptide from total cellular extracts of both HeLa and MDBK cells . The viral nucleoprotein was coimmunoprecipitated from influenza A/WSN/33 virus-infected MDBK cells by anti-NPI-1 sera, demonstrating an interaction of these two proteins in infected cells . Similarly, NPI-1 was coimmunoprecipitated from MDBK cells by anti-NP sera . These experiments suggest that NPI-1 plays a role during influenza virus replication.

Biochemistry, 1995 Jan 10, 34(1), 163 - 71
Structural studies of the roles of residues 82 and 85 at the interactive face of cytochrome c; Lo TP et al.; A combination of structural, functional, and mutagenic experiments has been used to study the roles of the invariant Phe82 and highly conserved Leu85 residues in cytochrome c, especially with respect to the complexation interface with electron transfer partners and maintenance of the hydrophobic heme pocket . Structural analyses show that the F82Y, L85A, and F82Y/L85A mutant proteins all retain the characteristic cytochrome c fold, but that conformational alterations are introduced in the direct vicinity of the mutation sites . In particular, the additional hydroxyl group of Tyr82 is in direct spatial conflict with the side chain of Leu85 in the F82Y mutant protein, leading to rotation of the side chain of Tyr82 out toward the protein surface . This strain is relieved in the F82Y/L85A mutant protein where the phenyl ring of Tyr82 is accommodated in a conformation comparable to that of the phenylalanine normally present at this location . In addition, the available space vacated by the replacement of Leu85 with an alanine allows for the inclusion of two new internal water molecules, one of which is bound to Tyr82 and the other to Arg13 . In contrast, in the L85A mutant protein, no internal water molecules are observed in this exclusively hydrophobic pocket, which is partially filled by shifts in nearby side chains . Overall, the conformational changes observed result from the optimization of side chain packing to reflect the spatial requirements of new side chains, the minimization of both vacant internal space and the solvent exposure of hydrophobic groups, and the attainment of maximal hydrogen bonding between available polar groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1995 Jan 6, 246(1), 80 - 90
A ste12 allele having a differential effect on a versus alpha cells; La Roche SD et al.; The transcriptional activator Ste12p is a key component of the yeast pheromone response pathway: phosphorylated as a consequence of signal transduction, it activates transcription of genes that promote mating and the subsequent fusion of the two cell types a and alpha . Activation by Ste12p requires three types of protein-protein interaction between DNA-binding activator proteins: (1) Ste12p by itself can induce non-cell-type-specific genes involved in mating; (2) cooperation of the transactivator Mcm1p with Ste12p induces a-specific genes; and (3) formation of a complex of the activator proteins Mcm1p and alpha 1 (a transcriptional activator of alpha-specific genes) with Ste12p is believed to induce alpha-specific genes . We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of alpha-specific genes . ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on mat alpha 2 mutant cells . In mat alpha 2 cells, where due to the lack of repressor, alpha 2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression . Thus, mat alpha 2 ste12-T50 cells mate like a cells . Additional ste12 mutants that confer the a mating phenotype on mat alpha 2 cells have also been isolated.

J Biol Chem, 1995 Jan 6, 270(1), 494 - 502
Requirement of tyrosine- and serine/threonine kinases in the transcriptional activation of the mammalian grp78/BiP promoter by thapsigargin; Cao X et al.; Depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin (Tg) in mammalian cells induces a set of ER protein genes known as the glucose-regulated proteins . Recently, IRE1p, a transmembrane protein postulated to have a serine/threonine kinase activity, has been identified as required for the induction of ER resident proteins genes in Saccharomyces cerevisiae . To investigate whether IRE1p can stimulate mammalian grp transcription, a stable Chinese hamster ovary cell line containing amplified copies of IRE1p has been created . The IRE1p expressing transfectants exhibited a modest (2-fold) enhancement of both the basal and Tg induced level of grp78 and grp94, two coordinately regulated grp genes . Using okadaic acid as a specific inhibitor for the endogenous serine/threonine protein phosphatase activities, a mild (2-fold) stimulative effect was observed for Tg induction of grp78 transcription . The okadaic acid potentiating effect requires a 50-base pair region in the vicinity of the grp78 TATA element . In contrast, the transcriptional activation of grp78 by Tg is almost totally eliminated by genistein, a tyrosine kinase inhibitor . The grp core, the C3 and C1 elements which are major Tg response elements of the rat grp78 promoter, are also major targets of the inhibitive effects of genistein.

Mol Gen Genet, 1995 Jan 6, 246(1), 56 - 64
Heat shock protein HSP60 can alleviate the phenotype of mitochondrial RNA-deficient temperature-sensitive mna2 pet mutants; Sanyal A et al.; mna2, which belongs to the class I temperature-sensitive pet mutants that lose mitochondrial (mt)RNA at restrictive temperature, was shown by complementation and sequence determination to correspond to the gene coding for HSP60 . Both mna2-1 and mna2-2, the two available alleles of mna2, have conservative single amino acid substitutions in the HSP60 gene . Valine substitutes for an alanine (position 47) in mna2-1, and an isoleucine substitutes for a valine (position 77) in mna2-2 . These substitutions result in defects in respiration and in steady-state mtRNA accumulation . Wild-type hsp60 alleviates the mtRNA phenotype completely, while partially relieving the respiratory deficiency.

Nature, 1995 Jan 5, 373(6509), 78 - 81
Role of a ubiquitin-conjugating enzyme in degradation of S- and M-phase cyclins; Seufert W et al.; Cell cycle progression in eukaryotes is controlled by the p34cdc2/CDC28 protein kinase and its short-lived, phase-specific regulatory subunits called cyclins . In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system . Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9 . Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA . In ubc9 mutants both CLB5, an S-phase cyclin, and CLB2, an M-phase cyclin, are stabilized . In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage . Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysis.

EMBO J, 1995 Jan 3, 14(1), 76 - 87
Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p; Grandi P et al.; Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide . In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p) . Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half . The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles . Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.

EMBO J, 1995 Jan 3, 14(1), 124 - 31
An inhibitor domain in c-Fos regulates activation domains containing the HOB1 motif; Brown HJ et al.; The c-Fos protein has three activation modules at its C-terminus, two of which contain motifs (HOB1 and HOB2) which are also present in the activation domains of c-Jun . Here we show the existence of two additional activation modules at the N-terminus of c-Fos, one of which contains a second HOB1 motif (HOB1-N) . The N-terminus also contains an inhibitor domain (ID1) which silences HOB1 activity . GAL4 fusion experiments showed that ID1 can specifically silence HOB1-containing activation domains from c-Fos or c-Jun when linked in cis, but will not affect other distinct activation domains . The c-Fos related protein, FosB, also contains an inhibitor domain . Mutagenic and deletion analyses identify an inhibitor motif (IM1) conserved between c-Fos and FosB, which is required for inhibitor function . Mutagenesis of IM1 enhances the ability of c-Fos to activate an AP1 bearing promoter . Finally, squelching experiments suggest that c-Fos ID1 binds a limiting protein involved in inhibition . These results demonstrate the existence of a new class of inhibitor domain within transcriptional activators, which acts in a sequence specific manner to inhibit a subset of activation domains.

Proc Natl Acad Sci U S A, 1995 Jan 3, 92(1), 165 - 9
Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique; Wang YK et al.; Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin . FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies . The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence . We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules . Ordered maps are then constructed by noting fragment order and size, using several optically based techniques . Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules . Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.

Med Arh, 1995, 49(1-2), 9 - 12
Nonspecific immunity in diabetes: hyperglycemia decreases phagocytic activity of leukocytes in diabetic patients; Jakelic J et al.; Phagocytic activity of leukocytes in blood was examined in 70 patients with diabetes mellitus . 40 of them had insulin-dependent diabetes--(IDDM) or type I, while there were 30 patients with noninsulin-dependent diabetes (NIDDM) or type II . Phagocytic activity of leukocytes was determined by quantitative method of ingestion, on the principle of quantifying phagocytized fungi (Saccharomyces cerevisiae) and free phagocytes (nonphagocytizing leukocytes) by means of a phase-contrast microscope . The data have been statistically processed by the Student t-test and variance analysis test (Duncan test) . The index of phagocytosis amounted to 3.2 +/- 0.77 in diabetic patients, while it was 3.47 +/- 0.29 in healthy examinees from the control group, thus yielding a statistically significant difference, p < 0.05 . Out of all examined parameters (blood glucose, glycosylized hemoglobin, mean glucose value) phagocytic activity of leukocytes showed a statistically significant correlation with mean value of glucose in blood . Patients with mean glucose value higher than 12 mmol/l showed a significantly lower index of phagocytosis (2.9 +/- 0.84) than patients whose mean glucose value was lower than 12 mmol/l (3.5 +/- 0.59), p < 0.05 . Neither age and sex of the patients nor chronic complications caused by diabetes affected the phagocytic activity of leukocytes in diabetic patients . There was no significant difference in the phagocytic activity of leukocytes between patients with IDDM and those with NIDDM.

J Inflamm, 1995-96, 47(1-2), 61 - 6
Identification of p55 tumor necrosis factor receptor-associated proteins that couple to signaling pathways not initiated by the death domain; Adam D et al.; The 55 kDa receptor for tumor necrosis factor (TNF-R55) has become a paradigm for membrane receptors that are devoid of intrinsic enzymatic activity . The initiation of intracellular signaling events observed in TNF-stimulated cells appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55 . By use of TNF-R55 deletion mutants, we have defined a novel domain of TNF-R55 (NSD) distinct from the death domain which is specifically required for activation of neutral sphingomyelinase (N-SMase) . In addition, using the yeast interaction trap system, we have identified one protein (FAN) that binds to the NSD and mediates activation of N-SMase as well as seventeen other potential interaction partners of TNF-R55 . These candidate interactors include the adaptor protein Grb2-linking TNF-R55 to the Ras pathway-as well as the enzyme phosphoglycerate mutase, suggesting a role of TNF-R55 in glycolysis/ energy metabolism of cells.

Mol Biol Rep, 1995-96, 22(2-3), 69 - 73
RNase MRP and rRNA processing; Lindahl L et al.; RNase MRP is a ribonucleoprotein enzyme with a structure similar to RNase P . It is required for normal processing of precursor rRNA, cleaving it in the Internal Transcribed Spacer 1.

Cell Mol Biol Res, 1995, 41(5), 435 - 40
Possible involvement of the mouse Grg protein in transcription; Mallo M et al.; The mouse Grg gene encodes a 197 amino acid nuclear protein homologous to the amino-terminal domain of the product of the groucho (gro) gene of the Drosophila Enhancer of split complex . Recent work has suggested that the gro protein functions as a transcriptional corepressor during Drosophila development . We therefore examined possible roles of the mouse Grg protein in DNA binding and in vitro transcription . No sequence-specific DNA binding activity was detected by polymerase chain reaction-DNA binding site selection nor was the glutamine-rich Grg protein capable of acting as an activation domain in an in vivo transactivation assay . However, depletion of Grg protein from HeLa nuclear extracts inhibited the in vitro transcription activity of the extracts . We suggest that Grg protein may interact with components of the basal transcription machinery.

Acta Biochim Pol, 1995, 42(4), 505 - 8
Fungal glycoproteins and their biosynthetic pathway as potential targets for antifungal agents; Tanner W et al.; The yeast cell wall as a good antifungal target is discussed in general . More specifically the reaction, catalyzed by Dol-P-Man: protein O-D-mannosyltransferase is proposed as a new potential target . Six genes responsible for this endoplasmic reticulum-localized reaction have been cloned and characterized so far . Triple disruptions of these genes are either lethal or the corresponding cells have to be osmotically stabilized to survive . No inhibitors of this reaction are as yet known.

Acta Biochim Pol, 1995, 42(4), 465 - 79
Ergosterol biosynthesis inhibition: a target for antifungal agents; Barrett-Bee K et al.; The isoprenoid sterols play a crucial role in the viability of all fungi; those unable to synthesise ergosterol because of inhibition, growth conditions or mutation must take it up from the environment . A range of compound types have been discovered which interfere with the biosynthetic pathway from acetate to ergosterol and these compounds have antifungal actions . Inhibition of several of the steps has yielded agents which have been used with great success as medical and agrochemical agents . The most important biosynthetic steps that have been exploited are inhibition of squalene epoxidase, (the allylamines and tolnaftate) C14 demethylation (the azoles), delta 7,8 isomerase and delta 14 reductase which are inhibited by the morpholines . Recent research has shown that inhibition of C24 methyltransferase and C4 demethylation also yield antifungal agents . Combination studies demonstrate that synergy between agents of different types can be measured . Fungicidal effects were observed when a combination of two fungistatic agents was used.

Crit Rev Eukaryot Gene Expr, 1995, 5(2), 127 - 56
Cyclins, cyclin-dependent kinases and cdk inhibitors: implications in cell cycle control and cancer; MacLachlan TK et al.; A significant portion of cell scientific literature published is dedicated to describing the cloning, the link to cancer, or the characterization of proteins involved in the progression of the cell cycle . With this abundance of information, the cascading pathways of molecular events that occur in the cell cycle are proving to be exceedingly complicated . Originally, the sole regulator of the fission yeast cells division cycle, cdc2, was thought to also regulate mammalian cell cycles in the same manner . However, mammalian cdc2 has now been joined by seven well-characterized relatives acting at distinct points in the cell cycle . These kinases are activated by larger proteins called cyclins, named with respect to their cyclical expression and degradation . Therefore, the catalytic subunits of these complexes are named cyclin-dependent kinases (cdks) . In the event that the cell must stop normal cycling behavior, a number of cdk inhibitors, which have only begun to be characterized, function in inhibiting the kinase ability of cdks, among other nonproliferative acts . The external environment manipulates cellular proliferation and differentiation by stimulating or inhibiting certain signal transduction pathways . However, each component of the cell cycle machinery, as they are the final executors in cell division, has the potential to elicit or to contribute to a neoplastic phenotype . This review focuses on the characterization of each member of the cell cycle protein family and also addresses the potential role each plays in cancer.

Nucleic Acids Symp Ser, 1995, (34), 143 - 4
DNA recognition by peptide oligomers; Yamane J et al.; It has been demonstrated that coupling a host-guest complex formation onto the DNA binding ability of short peptides gave raise a cooperative DNA binding by short peptide dimers . This strategy was extend to study the DNA binding by oligomers of short peptide . An amino acid bearing the adamantyl group was incorporated at the N-terminal of a peptide derived from the basic region of a yeast transcription activator GCN4, and beta-cyclodextrin was attached at the C-terminal cysteine residue in the same peptide chain . DNA binding of the peptide with both host and guest molecules to tandemly repeated DNA sequences was studied by gel shift assay.

Nucleic Acids Symp Ser, 1995, (34), 141 - 2
Sequence-specific DNA recognition by peptide heterodimers; Sawada M et al.; The DNA binding motif rich in basic amino acid residues is used by a variety of DNA binding proteins to recognize specific DNA sequences . We report here a sequence-specific DNA recognition by peptide heterodimers derived from the basic leucine zipper proteins . Specific hetero-dimerization is controlled by an artificial dimerization module consisting of a host-guest inclusion complex . Efficiency of the sequence discrimination by the peptide dimers depends on a stability of the half-matched binding complex . However, structures of the matched and half-matched binding complexes differ from one another.

Annu Rev Genet, 1995, 29, 423 - 44
Meiotic recombination hotspots; Lichten M et al.; Meiotic recombination occurs more frequently in some regions of the eukaryotic genome than in others, with variations of several orders of magnitude observed in frequencies of meiotic exchange per unit physical distance . This article reviews what is known abut meiotic recombination hotspots loci, or regions that display a greater than average frequency of meiotic exchange . Hotspots have been most intensively studied in Saccharomyces cerevisiae, which is a major focus of this article . Also reviewed is the current state of knowledge regarding hotspots in other fungi, in maize, in nematodes, in mice, and in humans.

Annu Rev Cell Dev Biol, 1995, 11, 677 - 706
COPs regulating membrane traffic; Kreis TE et al.; Cytosolic coat proteins (COPs) regulate membrane traffic in eukaryotic cells . Three classes of coat protein complexes have so far been identified: clathrin and its adaptor proteins, coatomer (COPI), and COPII . Coatomer (composed of seven different subunits) and ADP-ribosylation factor (ARF), which form the COPI coat, are required for budding of coated vesicles from membranes . COPI has been implicated in several steps of transport from the intermediate compartment to the cis-Golgi network, through cisternae of the Golgi stack, and is essential for retrieval to the endoplasmic reticulum (ER) of membrane proteins containing the carboxy-terminal dilysine ER-retention motif . A family of structurally and functionally related COPs may regulate all membrane traffic steps in eukaryotic cells.

Annu Rev Cell Dev Biol, 1995, 11, 633 - 75
Unconventional myosins; Mooseker MS et al.; Myosins are molecular motors that upon interaction with actin filaments convert energy from ATP hydrolysis into mechanical force . Evidence has emerged for the existence of a large, widely expressed and evolutionarily ancient superfamily of myosin genes . In addition to the well-catheterized conventional, filament-forming, two-headed myosin-II of muscle and nonmuscle cells, at least ten additional classes of myosins have been identified . In vertebrates, at least seven of the eleven classes are expressed, and many myosins can be expressed in a single cell type . This review summarizes known structural and functional features of these novel unconventional myosins.

Annu Rev Cell Dev Biol, 1995, 11, 519 - 48
Silencing and heritable domains of gene expression; Loo S et al.; Silencing is a process that assembles particular regions of eukaryotic chromosomes into transcriptionally inactive chromatin structures . Silencing involves specialized regulatory sites known as silencers and a combination of general DNA-binding proteins and proteins dedicated to silencing . In the yeast Saccharomyces cerevisiae, these proteins include transcription factors and the origin recognition complex (ORC) . Silencing has three recognizably separate phases: establishment, maintenance, and inheritance . At least some silencers are origins of replication, and the establishment of the silenced state requires an S phase-specific event . Once established, the silenced state is heritable, even in the absence of proteins required for its establishment . The silencing of mating-type genes bears many similarities to telomere position effects, and the two processes require many of the same proteins.

Annu Rev Cell Dev Biol, 1995, 11, 155 - 88
Protein import into the nucleus: an integrated view; Hicks GR et al.; The directed movement of macromolecules into and out of the nucleus is a fundamental process in eukaryotes and occurs through the nuclear pore complex (NPC) . A diverse array of molecules are transported across the nuclear envelope including proteins, mRNAs, tRNAs, snRNP complexes, ribosomal subunits, and in specialized cases, DNA . The structural and functional differences between these molecules point to the mechanistic complexity of NPCs and other components of the nuclear transport apparatus . This machinery must not only recognize within transported molecules specific targeting signals that differ between proteins, RNA, and RNA/protein complexes, it must translocate these molecules across the nuclear envelope . Additional levels of complexity are necessary because molecules such as proteins may continually undergo bidirectional transport across the envelope . Beyond these basic functions, the nuclear transport apparatus is regulated at the level of individual substrates and at more global levels such as coupling to cell cycle regression.

Cell Motil Cytoskeleton, 1995, 32(2), 133 - 5
Functional dissection of the dynein motor domain; Eshel D; The highly conserved lysine residue in the putative hydrolytic ATP-binding motif of the yeast cytoplasmic dynein heavy chain was replaced with leucine . The mutation was generated by a two-stage transformation method designed for genomic site-directed mutagenesis . Preliminary observations show that the effects of this alteration on the cellular roles of dynein are indistinguishable from those of a disruption mutation in which the entire motor domain is not expressed.

J Inflamm, 1995, 45(3), 143 - 51
Regulation of GRO alpha production in human granulocytes; Gasperini S et al.; GRO alpha, a member of the chemokine superfamily, exerts potent stimulatory actions on granulocytes . In this report, we show that activated human polymorphonuclear leukocytes (PMN) are able to produce significant amounts of GRO alpha . Lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and yeast particles opsonized with IgG (Y-IgG) had the ability to induce GRO alpha release, with Y-IgG being the most potent stimulus . The extracellular production of GRO alpha was also modulated by both interferon-gamma (IFN gamma) and interleukin-10 (IL-10) . IFN gamma significantly inhibited the production of GRO alpha by PMN stimulated for 2 hr with LPS, TNF alpha, or Y-IgG, but potentiated the production of GRO alpha in cells stimulated for 18 hr with LPS and TNF alpha . IL-10 moderately suppressed the Y-IgG-induced production of GRO alpha, but strongly inhibited the action of LPS and potentiated the effect of TNF alpha . As revealed by Northern blot analysis, the extracellular production of GRO alpha under the experimental conditions used did not always correlate with parallel changes at the level GRO alpha mRNA expression, suggesting that production of GRO alpha by PMN might be regulated at post-transcriptional, translational, or post-translational level . These findings identify a novel biological function of PMN, likely involved in the modulation of the acute inflammatory response.

Biochimie, 1995, 77(7-8), 516 - 30
Flavocytochrome b2-cytochrome c interactions: the electron transfer reaction revisited; Capeillere-Blandin C; This review is concerned with the kinetics and mechanism of electron transfer processes which occur intermolecularly between reduced flavocytochrome b2 and cytochrome c molecules within an encounter complex . Analyses are given of previous reports which aimed at describing the formation of stable complexes obtained at low ionic strength in solution and in the crystalline state with a binding stoichiometry of 1 to 1 heme ratio . Relevant data allow to define the respective role of flavin and heme b2 in the electron transfer towards cytochrome c and give a description of the recognition areas on the two redox partners . The paper also refers to a recent computer model of their postulated interactions as based on the three-dimensional structure of the Saccharomyces cerevisiae single molecules . Special emphasis is given to rapid kinetic investigations of the electron transfer reaction between Hansenula anomala flavocytochrome b2 and cytochrome c studied as a function of concentration, ionic strength and temperature . Data showed that reaction rates were modulated by ionic strength, reaching a saturation behaviour at low ionic strength . In the present paper the temperature effects on Kd and kET have been re-examined . Thermodynamic analysis of the dissociation constant points out the importance of hydrophobic interactions in the complex formation . Analysis of the variations of rate constants in terms of semiclassical theory of electron-transfer reaction yields values of 1.12 eV for the reorganization energy and 0.05 cm-1 for the electronic coupling factor . Interpretation of the electronic coupling in terms of through-bond and/or through-space pathways takes into account the hypothetical model proposed for the binary complex . The functional implications of this model in the electron transfer reaction are discussed . Finally the existence of a conformational equilibrium between the initial binding complex and the complex from which electron transfer occurs is considered.

Cell Mol Biol Res, 1995, 41(2), 85 - 95
NFI/X proteins: a class of NFI family of transcription factors with positive and negative regulatory domains; Nebl G et al.; NFI is a family of transcription factors that binds discrete nucleotide sequence and regulates transcriptional activity of genes . Transactivation occurs through proline-rich sequences . Among the genes whose transcriptional activity is regulated are those expressed in a cell-type specific manner . This suggests the possible existence of NFI proteins with distinct properties . With the use of the polymerase chain reaction technique, we have cloned and analyzed the activity of three spliced variants of NFI (class X) genes in murine cells . Expression vectors containing the regulatory regions of these proteins fused to the DNA binding domain of the yeast transcription factor GAL4 helped identify both negative and positive regulatory domains . In the context of the whole NFI/X proteins, spliced variants lacking the positive regulatory sequences functioned as repressors, whereas those containing both regulatory domains functioned as weak activators . With the identification of the negative domain in the NFI/X proteins, we demonstrate here a novel regulatory feature of these proteins in positive and negative modulation of gene expression.

Mol Biol Rep, 1995, 21(2), 87 - 94
The inhibitory domain in the Oct-2 transcription factor represses gene activity in a cell type-specific and promoter-independent manner; Lillycrop KA et al.; The Oct-2 transcription factor contains an N-terminal inhibitory domain which can act to inhibit promoter activity when linked to either its corresponding DNA-binding POU domain or the heterologous DNA binding domain of the yeast transcription factor GAL4 . This inhibitory effect is independent of the number of DNA binding sites or their context in the target promoter . In contrast the effect is cell type-specific and can be relieved by over-expression of the isolated inhibitory domain in the absence of a DNA binding domain . These results suggest that the inhibitory domain acts by decreasing the activity of the basal transcriptional complex but that it operates indirectly by recruiting a second cell type-specific factor to the promoter which then interacts with the basal complex decreasing its activity.

Methods Enzymol, 1995, 255, 331 - 42
Ras-Raf interaction: two-hybrid analysis; Vojtek AB et al.; The identification of proteins mediating Ras effects, such as the serine/threonine kinases of the Raf family, has advanced our understanding of how signals are transmitted from the extracellular milieu to the nucleus . The modified two-hybrid system has proved to be a powerful tool for identifying specific protein interactions, such as those between Ras and Raf . We hope that the insight gained from the Ras screen, as well as insights from other two hybrid screens, will prove valuable in the application of this system to other enigmatic questions in biology.

J Immunol, 1995 Jan 1, 154(1), 268 - 80
Alteration of pulmonary macrophage function by respiratory syncytial virus infection in vitro; Franke-Ullmann G et al.; Alveolar macrophages (AL) are the first line of defense against inhaled pathogens and are exposed to virus during the course of a respiratory syncytial virus (RSV) infection . Interference of virus with alveolar macrophage functions may contribute to the risk of acquiring secondary bacterial infections during or after respiratory tract infections with RSV or other viral agents . We studied whether murine AL get infected with RSV and whether they support viral replication in vitro . In addition, the effects of RSV on microbicidal and on immunoregulatory functions were examined . Only a subpopulation of AL expressed viral F proteins after exposure of these cells to RSV . Infected AL released only small amounts of infectious virus into the supernatant . The extent of virus replication in AL seemed to be dependent in part on the amount of IFN induced by the virus, as has been demonstrated by infection of lung tissue macrophages and AL in vitro . In general, RSV infection of pulmonary macrophages appeared to be abortive . Nevertheless, release of reactive oxygen intermediates, phagocytosis, and killing of protozoa were reduced in RSV-infected AL in comparison to noninfected AL . In contrast, RSV stimulated secretion of TNF-alpha, IL-1, and IL-6 in an infectious-dose dependent manner . Along with the increased cytokine release, accessory functions of AL were increased after RSV exposure . Thus, exposure of AL to RSV appeared to stimulate their immunoregulatory functions, whereas the microbicidal activity of these cells seemed to be severely diminished.

Cancer Chemother Pharmacol, 1995, 35(6), 519 - 26
Effects of hexadecylphosphocholine on fatty acid metabolism: relation to cytotoxicity; Goppelt-Struebe M et al.; The cytotoxicity of the antineoplastic drug hexadecylphosphocholine (HePC) was determined in a human monocytic tumor cell line, THP1, and in primary cultures of rat mesangial cells . Both cell types were susceptible to HePC toxicity, the concentrations producing 50% inhibition of cell viability (LD50 values) being 7 micrograms/ml for THP1 cells and 19 micrograms/ml for mesangial cells . The degree of toxicity was dependent on the culture conditions . In the absence of serum, HePC was highly toxic independent of cell proliferation . As a potential molecular mechanism, the effect of HePC on long-chain fatty acyl metabolism was investigated . HePC had no effect on fatty acid incorporation into cellular lipids or on release of fatty acids from lipid stores . The distribution of labeled fatty acids, however, was shifted from the phospholipid fraction to the triacylglycerol fraction . This effect was in accordance with an inhibition of the activity of the reacylating enzyme lysophosphatide acyltransferase . There was, however, no correlation between the interference with fatty acid distribution and HePC cytotoxicity in vitro . The data argue against interference with membrane fatty acid metabolism as a necessary prerequisite of HePC toxicity, the mechanism of which remains to be elucidated.

Endocrinology, 1995 Jan, 136(1), 357 - 60
Distribution of the Kexin family proteases in pancreatic islets: PACE4C is specifically expressed in B cells of pancreatic islets; Nagamune H et al.; The distribution of Kexin family proteases in adult rat pancreatic islets was investigated by immunohistochemical means using a series of specific antibodies specific for PC1, PC2, PC6, Furin, PACE4A and a recently identified member of the Kexin family, PACE4C . PACE4C expression was limited to B cells of the pancreatic islets . PC2 was found in A and in some D cells more than in B cells and PC1 was evident only in B cells . Furin and PC6 were weakly and evenly expressed in the entire islet . PACE4A was hardly found in the islets . These findings indicated that individual Kexin family proteases are uniquely distributed in the islets and suggested that these proteases share roles in these cells as follows: PC2 is involved in the peptide hormone precursor processing in A cells and in D cells, and PACE4C, PC1 and PC2 (mainly PACE4C and PC1) are responsible for the processing event(s) specific to B cells.

Mol Cell Biol, 1995 Jan, 15(1), 298 - 304
Pulling the ribosome out of frame by +1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA; Pande S et al.; Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation . A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tRNA occupying the P site during the pause . The special nature of the peptidyl-tRNA reflects its ability to slip +1 on the mRNA or to facilitate binding of an incoming aminoacyl-tRNA out of frame in the A site . This second mechanism suggested that in some cases the first +1 frame tRNA could have an active role in frameshifting . We found that overproducing this tRNA can drive frameshifting, surprisingly regardless of whether frameshifting occurs by peptidyl-tRNA slippage or out-of-frame binding of aminoacyl-tRNA . This finding suggests that in both cases, the shift in reading frame occurs coincident with formation of a cognate codon-anticodon interaction in the shifted frame.

Genomics, 1995 Jan 1, 25(1), 274 - 8
Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23; Okumura K et al.; Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells . We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits {p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)} using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Genomics, 1995 Jan 1, 25(1), 1 - 8
MATS: a rapid and efficient method for the development of microsatellite markers from YACs; Chen H et al.; In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs) . This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome . Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis . Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences . Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol . Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites) . These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval . In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.

J Struct Biol, 1995 Jan-Feb, 114(1), 60 - 6
A novel method for transfer of two-dimensional crystals from the air/water interface to specimen grids . EM sample preparation/lipid-layer crystallization; Asturias FJ et al.; Transfer of two-dimensional (2-D) crystals formed on lipid layers by suspension from a wire loop is described . This method gives better recovery and better preservation of 2-D crystals than attained in the past . The method has been applied to crystals of yeast RNA polymerase II to enable their analysis in the frozen hydrated state.

Yeast, 1995 Jan, 11(1), 43 - 52
In vivo evidence for non-universal usage of the codon CUG in Candida maltosa; Sugiyama H et al.; An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds . However, functional expression of heterologous genes tested for this purpose had not been successful in several cases . On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C . cylindracea . The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C . maltosa . In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C . maltosa . This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C . maltosa containing a CTG codon was expressed in C . maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene of C . albicans could be isolated from the genome of C . maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C . maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation . The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.

Mol Endocrinol, 1995 Jan, 9(1), 72 - 85
Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors; Seol W et al.; We have used a yeast genetic system to isolate cDNAs encoding proteins that specifically interact with the ligand-binding domain of human retinoid X receptor-alpha (RXR alpha) . A number encoded portions of two known RXR heterodimer partners, the retinoic acid receptor (RAR) and the peroxisome proliferator activated receptor . Of four additional RXR-interacting proteins (RIPs) selected for further study two, RIP14 and RIP15, are previously unidentified orphan members of the nuclear receptor superfamily . Two others, RIP110 and RIP13, do not show significant similarities to previously reported proteins . RIP110 interacts with LexA-RXR only in yeast cells grown in the presence of the RXR ligand 9-cis-RA, while the interaction of the four receptor superfamily members and RIP13 is unaffected by the presence or absence of 9-cis-RA . RIP110 and RIP13 also interact in yeast with several other members of the receptor superfamily, but RIP14 and RIP15 interact only with RXR . Analysis of larger cDNA clones demonstrates that there are at least two isoforms of RIP14 that differ in the N-terminal (A and B) and hinge (D) domains . Northern blot analysis indicates that RIP14 is expressed specifically in liver and kidney, while RIP15 is expressed in every tissue tested . Both RIP14 and 15 bind as heterodimers with RXR to the RA response element (RARE) from the promoter of the RAR beta 2 isoform (the beta RARE), and RIP14 and RXR heterodimers also bind the ecdysone response element from the Drosophila heat shock protein 27 promoter . Both heterodimers also bind to several synthetic RAREs and other elements . In cotransfections, neither RIP14 nor RIP15 trans-activates a reporter containing multiple copies of the beta RARE under any of a variety of conditions, suggesting that their activities are dependent on the binding of as yet unidentified specific ligands or on activation by other processes.

Mol Endocrinol, 1995 Jan, 9(1), 34 - 43
Enhancement of human estrogen receptor activity by SPT6: a potential coactivator; Baniahmad C et al.; The conserved nature of the transcriptional machinery between yeast and higher eukaryotes makes the yeast system suitable to genetically dissect the signal transduction pathway of steroid hormone receptors . This report describes the yeast protein, SPT6, which modulates the transcriptional activity of the human estrogen receptor (hER) by affecting the C-terminal activation domain . It is demonstrated that SPT6 is able to potentiate hER activity in yeast and also in mammalian cells in vivo . SPT6 interacts specifically with the hormone-binding domain of hER in vivo . The in vivo studies are substantiated by specific protein-protein interactions between SPT6 and the hormone-binding domain of hER in vitro . Therefore, the data suggest that the SPT6 protein may be involved in signal transmission of ER by acting as a coactivator.

Mol Biol Cell, 1995 Jan, 6(1), 59 - 70
Control of RAS mRNA level by the mevalonate pathway; Dimster-Denk D et al.; The ability of Ras proteins to initiate eukaryotic cell proliferation requires the post-translational attachment of a farnesyl group, an isoprenoid lipid moiety derived from mevalonate, to the carboxyl-terminus of the protein . This modification is essential for the subsequent processing and intracellular targeting of the Ras protein . Here we report that mevalonate is also required for the efficient synthesis of Ras proteins in Saccharomyces cerevisiae . Depletion of intracellular mevalonate resulted in decreased steady-state levels of Ras1p and Ras2p, an effect that was mediated at the level of mRNA accumulation . The sequences controlling the response of RAS2 mRNA level to mevalonate availability, mapped to the coding region of the RAS2 gene . Mevalonate starvation also had a significant effect on the expression of some, but not all, genes encoding prenylated proteins . The regulatory effect on RAS2 mRNA did not require a functional farnesyl transferase . These results uncover a novel regulatory role for mevalonate-derived products and expand the potential for inhibitors of mevalonate metabolism as anti-cancer agents.

Biol Pharm Bull, 1995 Jan, 18(1), 195 - 7
Molecular cloning of cDNA encoding rat 2,3-oxidosqualene: lanosterol cyclase; Kusano M et al.; A cDNA encoding rat 2,3-oxidosqualene: lanosterol cyclase, the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis, was cloned by extensive application of PCRs . Oligonucleotide primers were designed in sense and anti-sense directions corresponding to internal peptide sequences of purified enzyme . Successive PCRs with all possible combinations of these primers yielded a 178-bp fragment, and based on its sequence full nucleotide sequence of cDNA was obtained by a "rapid amplification of cDNA ends" (RACE) method.

Gene Expr, 1995, 4(3), 111 - 24
E1BF/Ku interacts physically and functionally with the core promoter binding factor CPBF and promotes the basal transcription of rat and human ribosomal RNA genes; Niu H et al.; We have previously characterized an RNA polymerase (pol) I transcription factor, E1BF, from rat cells . This protein is immunologically related to Ku autoantigen and is required in pol-I directed transcription of rodent ribosomal RNA gene (rDNA) . Glycerol density gradient fractionation and in situ UV cross-linking analysis of the purified factor showed directly that it consists of a heterodimer of 85 and 72 kDa polypeptides . E1BF also interacted with the human core promoter and augmented transcription of human rDNA as much as fivefold in HeLa nuclear extract, whereas transcription from adenovirus major late promoter, CMV or SV40 early promoters by pol II and of U6 and 5S RNA genes by pol III were either unaffected or minimally inhibited by the antibodies . Purified rat E1BF partially restored the suppression of human rDNA transcription by anti-Ku antibodies . Immunoprecipitation of rat cell extract with the anti-Ku antibodies followed by SDS-PAGE of the precipitated proteins and Southwestern analysis showed that E1BF interacts with CPBF, a core promoter binding factor . When the majority of CPBF and E1BF was removed from the reaction mixture by preincubation with a core promoter oligo nucleotide fragment, rDNA transcription was severely impaired . Addition of exogenous CPBF or E1BF to such a reaction resulted in significant restoration of the transcription, whereas inclusion of both factors caused further enhancement of rDNA transcription . These data demonstrate that E1BF is a basal pol I transcription factor that interacts with a core promoter binding factor both physically and functionally, and that is not a general pol II or pol III transcription factor.

Chem Res Toxicol, 1995 Jan-Feb, 8(1), 136 - 42
Oxidation of benzo{a}pyrene by recombinant human cytochrome P450 enzymes; Bauer E et al.; The oxidation of benzo{a}pyrene (B{a}P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18 . Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume . The most active enzyme in all reactions was P450 1A1 . P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol . P450 2C enzymes and P450 2E1 formed relatively low amounts of all B{a}P products . Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B{a}P in tissues where it is present and inducible . In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute . Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol . 7,8-Benzoflavone stimulated the oxidation of B{a}P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2 . The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1 . The major 7,8-benzoflavone product appears to be the 5,6-oxide.

Allerg Immunol (Paris), 1995 Jan, 27(1), 7 - 10
{Respiratory allergies among symptomatic bakers and pastry cooks: initial results of a prevalence study}; Bataille A et al.; A survey was carried out on respiratory symptoms and skin prick response to common allergens, storage mite and occupational allergens . Among 178 symptomatics bakers and pastry workers from small businesses in western France, only 65 people underwent skin prick and specific-IgE . 12 (18%) workers were skin positive to at least one common or occupational allergens . The more often skin positive were D . Ptero . mite 36 (57%); Alpha amylase 23 (35%); wheat flour 17 (26%); saccharomyces cerevisiae 16 (25%); Ephestia 15 (24%) . The sensitivity of skin test was better than specific IgE for D . Ptero . Mite 36 (57%); and Alpha amylase 23 (35%) . The sensitivity of specific IgE was better than skin test for wheat flour 26 (45%) and rye flour 23 (40%) . Occurrence of skin positive to occupational allergen among symptomatics with rhinitis and asthma is much more frequent in workers with skin positive to common allergens (40/36) than in workers with skin negative (8/20) . Atopy must be regarded as an important predisposing factor for skin sensitisation to occupational allergens . We conclude in the necessity of a standardised allergologic exploration to be done in symptomatics bakers.

Plant Cell, 1995 Jan, 7(1), 85 - 103
A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif; Soni R et al.; A new family of three related cyclins has been identified in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins . Individual members show tissue-specific expression and are conserved in other plant species . They form a distinctive group of plant cyclins, which we named delta-type cyclins to indicate their similarities with mammalian D-type cyclins . The sequence relationships between delta and D cyclins include the N-terminal sequence LXCXE . This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein pRb . By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle . In support of this hypothesis, we show that, on restimulation of suspension-cultured cells, cyclin delta 3 is rapidly induced by the plant growth regulator cytokinin and cyclin delta 2 is induced by carbon source.

Crit Rev Biochem Mol Biol, 1995, 30(2), 121 - 64
Nucleo-mitochondrial interactions in mitochondrial gene expression; Grivell LA; All proteins encoded by mitochondrial DNA (mtDNA) are dependent on proteins encoded by nuclear genes for their synthesis and function . Recent developments in the identification of these genes and the elucidation of the roles their products play at various stages of mitochondrial gene expression are covered in this review, which focuses mainly on work with the yeast Saccharomyces cerevisiae . The high degree of evolutionary conservation of many cellular processes between this yeast and higher eukaryotes, the ease with which mitochondrial biogenesis can be manipulated both genetically and physiologically, and the fact that it will be the first organism for which a complete genomic sequence will be available within the next 2 to 3 years makes it the organism of choice for drawing up an inventory of all nuclear genes involved in mitochondrial biogenesis and for the identification of their counterparts in other organisms.

Methods Enzymol, 1995, 250, 235 - 51
Ras and a-factor converting enzyme; Ashby MN et al.; We have described several quantitative and qualitative assays that have been utilized to learn the basic properties of RACE and amphibian and mammalian counterparts . Owing to powerful genetic tractability, high specific activity, and an apparently well-conserved substrate specificity, yeast is an attractive organism in which to study RACE . Efforts are currently in progress to characterize the functional role of the endoproteolytic processing step of many essential proteins.

Methods Enzymol, 1995, 250, 169 - 86
Lipid-mediated a-factor interactions with artificial membranes; Epand RM et al.; Our understanding of the cellular export of a-factor and its interaction with the receptor do not yet allow for a description of the phenomena on a molecular level . Synthesis of a-factor analogs and biophysical studies of the lipopeptides in the presence of artificial membranes provide insights which can be analyzed with respect to the biological potency of the molecules . It is through the study of the interaction of the lipopeptides with membranes at varying levels of complexity that we will be able to develop a molecular description of the biological processes.

Int Rev Immunol, 1995, 12(2-4), 129 - 44
Molecular structure and function of autoantigens in systemic sclerosis; Lee B et al.; Autoantibodies in systemic sclerosis target a limited set of nuclear proteins, principally those of the nucleolus and RNA transcription complexes . These antibodies have proved helpful in diagnosis of this disease, and have been used extensively as probes of nuclear structure and function . Despite these advances, the events that initially trigger autoantibody production in systemic sclerosis are not yet known . While these ANA are not known to disrupt cellular processes by entering living cells, or to cause tissue injury (in contrast to SLE, where autoantibodies may mediate tissue damage), it seems likely that they do not merely represent epiphenomena of the disease . Rather, it is logical to assume that their origin is in some manner tied to etiology of systemic sclerosis, since they segregate by syndrome within the spectrum of this disease (for example, anti-kinetochore antibodies occur in limited cutaneous disease, and anti-topoisomerase I and anti-RNA polymerase antibodies occur in diffuse disease), and since they are distinct from the ANA found in other connective tissue diseases in their selectivity for the nucleolus and RNA polymerases.

Adv Biophys, 1995, 31, 93 - 100
A species-specific interaction of rad51 and rad52 proteins in eukaryotes; Ogawa T et al.; The structures and properties of the Rad51 and Rad52 proteins in eukaryotes are described . Both proteins form a complex and are responsible for recombination and repair reactions . The N-terminal region of the Rad51 protein interacts with the C-terminal region of the Rad52 protein . Species-specific interaction is probably essential for the functioning of these genes.

Biochimie, 1995, 77(1-2), 45 - 53
Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase; Rose AM et al.; Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway . Indirect immunofluorescence experiments using antibody to N2,N2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells . Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p . Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease . Cells overproducing Trm1p also concentrate it to the nuclear periphery . Thus, the site(s) of interaction of Trm1p are not easily saturable and are likely to be in excess to Trm1p . Trm1p is shared by mitochondria and the nucleus . Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme . Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.

Proc Int Conf Intell Syst Mol Biol, 1995, 3, 393 - 401
Neweyes: a system for comparing biological sequences using the running Karp-Rabin Greedy String-Tiling algorithm; Wise MJ; A system for aligning nucleotide or amino acid biosequences is described . The system, called Neweyes, employs a novel string matching algorithm . Running Karp-Rabin Greedy String Tiling (RKR-GST), which involves tiling one string with matching substrings of a second string . In practice, RKR-GST has a computational complexity that appears close to linear . With RKR-GST, Neweyes is able to detect transposed substrings or substrings of one biosequence that appears rearranged in a second sequence . Repeated substrings can also be detected . Neweyes also supports a form of matching-by-group that gives the effect of different amino acid mutation matrices . Neweyes can be used in a macro mode (searching a database for a list of biosequences that are similar to a given biosequence) or in a micro mode, where two biosequences are compared and more detailed output formats are available.

Proc Int Conf Intell Syst Mol Biol, 1995, 3, 197 - 205
Computer tool FUNSITE for analysis of eukaryotic regulatory genomic sequences; Kel AE et al.; We present the computer tool FUNSITE for description and analysis of regulatory sequences of eukaryotic genomes . The tool consists of the following main parts: 1) An integrated database for genomic regulatory sequences . The integrated database was designed on the basis of the databases TRANSFAC (Wingender 1994) and TRRD (Kel et al . 1995) that are currently under development . The following functions are performed: i) linkage to the EMBL database; ii) preparing samples of definite types of functional sites with their flanking sequences; iii) preparing samples of promoter sequences; iv) preparing samples of transcription factors classified with regard to structural and functional features of DNA binding and activating domains, functional families of the factors, their tissue specificity and other functional features; v) access to data on mutual disposition of cis-elements within the regulatory regions . 2) The second component of FUNSITE tool is the set of programs for analysis of the structural organization of regulatory sequences: i) Program for revealing of potential transcription factors binding sites based on their consensi; ii) program for revealing of the potential binding sites using homology search with nucleotide sequences of real binding sites; iii) program for analysis of oligonucleotide context features which are characteristic of flank sequences of the binding sites; iv) program for design of recognition method for the functional sites based on generalized weight matrix; v) program for revealing potential composite elements . The results of analysis of the promoter sequences of eukaryotic genes with the FUNSITE are presented, too.

Nephrol Dial Transplant, 1995, 10(6), 847 - 54
Evaluation by histology, immunohistology and PCR of protocollized renal biopsies 1 week post-transplant in relation to subsequent rejection episodes; Kooijmans-Coutinho MF et al.; Renal biopsies were performed 1 week following renal transplantation at a time without clinical evidence of rejection in 43 patients (13 females, mean age 48 years range 18-60 and 30 males, mean age 43 years range 17-59 years) . Thirty-six biopsies were available for histological or immunohistochemical analysis . Immunohistochemical analyses were performed with monoclonal antibodies against leukocytes (CD45), monocytes (WT14), complement factor 3 (C3), T-cells (Leu4), T-cell receptor alpha beta and gamma delta, tumour necrosis factor alpha (TNF alpha), IL-2 receptor (IL2-R, TAC), intercellular adhesion molecule-1 (ICAM1) and HLA-DR . The slides were scored semiquantitatively with the observers having no knowledge of clinical or patient data . TNF alpha and IL-2R were also measured by quantative PCR . None of the studied parameters correlated to delayed graft function or graft loss . Histological analysis showed that both focal interstitial infiltrate (18/35) and tubular basement membrane disruption (11/35) were followed by a higher incidence of subsequent rejection (P = 0.03 and 0.02 respectively) . Also positivity for WT14 around tubuli (P = 0.02) was associated with subsequent occurrence of rejection . The intensity of staining of ICAM-1 on PTC as well as TAC on proximal tubular cells was associated with the number of subsequent rejection episodes . The association between the IL-2 receptor and subsequent rejection was also found applying PCR to the tissue specimens . We conclude that the presence of focal interstitial infiltrates and tubulitis in 1-week biopsies from well-functioning grafts carries an increased risk of subsequent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Motil Cytoskeleton, 1995, 31(4), 255 - 8
Identification of two new members of the tubulin family; Burns RG; Analysis of the delta- and epsilon-tubulin sequences indicates that they both consist of two structural domains of which the N-terminal domain can bind to alpha/beta heterodimers while the C-terminal domain probably binds to a non-tubulin protein . Both additional tubulins probably bind GTP but lack GTPase activity, while their synthesis requires the TCP1 chaperonine but is not autoregulated . Although these properties resemble those of gamma-tubulin, the low sequence identity (Table I) demonstrates that the gamma-, delta-, and epsilon-proteins should be classed as different members of the tubulin family . The identification of these additional members is unexpected . Examination of the cellular expression and distribution of the delta- and epsilon-tubulins, and whether other organisms contain homologous genes, may reveal further features of the eukaryotic cytoskeleton.

J Enzyme Inhib, 1995, 8(4), 255 - 9
Enolase and the arsonomethyl analogue of 2-phosphoglycerate; Chawla S et al.; (RS)-3-Arsono-2-(hydroxymethyl)propionic acid was synthesized by the action of alkaline arsenite on 3-bromo-2-(bromomethyl)propionic acid . It is a substrate for yeast enolase (EC 4.2.1.11) with a Km of 6.5 mM (for 2-phospho-D-glycerate Km = 0.08 mM) . The catalytic constant of the enzyme with the arsonomethyl analogue is 230 times lower than with 2-phosphoglycerate.

Mol Carcinog, 1995 Jan, 12(1), 42 - 9
Deletion analysis of protein kinase C inactivation by calphostin C; Rotenberg SA et al.; Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C . Both PKC-alpha (a Ca(2+)-dependent conventional isoform) and PKC-epsilon (a Ca(2+)-independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC . We therefore performed studies to identify a critical region in the regulatory domain of PKC-alpha required for inactivation by calphostin C . A series of N-terminal-truncation mutants of bovine PKC-alpha expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-alpha by 80-90% . This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140) was inactivated by only 20% . These findings imply that the aa sequence 92-140 is a structural determinant of PKC-alpha inactivation by calphostin C . This sequence contains one of the phorbol ester-binding sites (aa 102-144), which is highly conserved among most PKC isoforms including PKC-epsilon . In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity . These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor . The significance of the proposed proximity effect to calphostin C action is discussed.

Mutat Res, 1995 Jan, 336(1), 19 - 27
Genetic changes and bioassays in bleomycin- and phleomycin-treated cells, and their relationship to chromosomal breaks; Koy JF et al.; The recombinogenicity of damaged chromosomes in diploid Saccharomyces cerevisiae cells treated with bleomycin and structurally related phleomycin was measured, along with aneuploidy and mutation events . Phleomycin was substantially (up to 26-fold) more effective than bleomycin in producing genetic changes at all concentrations, even when colony-forming abilities of cells growing in the presence of bleomycin or phleomycin were similar . These results suggest that the DNA lesions produced by the two structurally related analogs could differ in their nature or frequency, or could be processed differently by the cells . Bioassays were developed and used to compare the cytotoxicities of freshly dissolved bleomycin and phleomycin with the cytotoxicities of lysates prepared from bleomycin- and phleomycin-treated cells . Unexpectedly, lysates prepared from bleomycin-treated cells were 1.5-3.5 times more cytotoxic than freshly dissolved bleomycin after 45-min treatments (3-33 x 10(-6) M) . In contrast, lysates prepared from phleomycin-treated cells were 3-38 times less cytotoxic than freshly dissolved phleomycin (0.5-6.4 x 10(-6) M) . Cytotoxicities of all lysates were higher after 36-h treatments than after 45-min treatments . At 3.3 x 10(-6) M, this increase was eightfold for bleomycin and 15-fold for phleomycin . Nevertheless, lysates from phleomycin-treated cells were considerably more cytotoxic than lysates from bleomycin-treated cells or freshly prepared bleomycin, consistent with the higher effectiveness of phleomycin than bleomycin in producing chromosomal breaks, genetic changes, and cell killing.

Mol Cell Biol, 1995 Jan, 15(1), 580 - 9
A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence; Xu B et al.; Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans . These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA . During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region . The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required . The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed . Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase . These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

Mol Cell Biol, 1995 Jan, 15(1), 217 - 26
Multiple molecular determinants for retrotransposition in a primer tRNA; Keeney JB et al.; Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA . Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion . The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis . The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood . We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process . This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis . We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely . Conversely, mutations in the anticodon region have only minimal effects on transposition . Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming . Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants . Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

Arch Virol, 1995, 140(11), 2067 - 73
Expression and analysis of the NS2 protein of influenza A virus; Ward AC et al.; Influenza NS2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter . The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus . Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1 . A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.

J Comput Biol, 1995 Spring, 2(1), 117 - 23
ORFs and genes: how strong a connection?
Fickett JW.
The length of an open reading frame (ORF) is one important piece of evidence often used in locating new genes, particularly in organisms where splicing is rare . However, there have been no systematic studies quantifying the degree of correlation between length of ORF, on the one hand, and likelihood of gene function, on the other . In this paper, techniques are derived to estimate the conditional probability of gene function, given ORF length, based on evidence both from the databases and from simulation . Several complete chromosomes of Saccharomyces cerevisiae have now been sequenced, and considerable effort is being expended on locating and characterizing the genes in these sequences . Thus, we illustrate the techniques for this organism.

Peptides, 1995, 16(5), 933 - 8
Production of bioactive enkephalin from the nonendocrine cell lines COS-7, NIH3T3, Ltk-, and C2C12; Takahashi K et al.; Enkephalin is synthesized from proenkephalin in neuroendocrine cells . For the attempt to induce nonneuroendocrine origin cells to produce enkephalin, we used a mammalian expression vector for fusion peptides, pMEproCT beta, in which a fused peptide is designed to be cleaved by a yeast Kex2-like endoprotease furin . Met-Enkephalin was expressed in four nonneuroendocrine cell lines: COS-7, C2C12, Ltk-, and NIH3T3 . The four cell lines produced a marked amount of Met-enkephalin, which appeared as a single peak on reverse-phase HPLC . Because transplantation of adrenal medullary cells to the subarachnoid space has been used to alleviate terminal cancer pain, and enkephalin appears to play a central role in relieving pain, this enkephalin expression vector may be useful for direct enkephalin expression in pericancerous tissues.

Cell, 1994 Dec 30, 79(7), 1199 - 207
Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum; Letourneur F et al.; Dilysine motifs in cytoplasmic domains of transmembrane proteins are signals for their continuous retrieval from the Golgi back to the endoplasmic reticulum (ER) . We describe a system to assess retrieval to the ER in yeast cells making use of a dilysine-tagged Ste2 protein . Whereas retrieval was unaffected in most sec mutants tested (sec7, sec12, sec13, sec16, sec17, sec18, sec19, sec22, and sec23), a defect in retrieval was observed in previously characterized coatomer mutants (sec21-1, sec27-1), as well as in newly isolated retrieval mutants (sec21-2, ret1-1) . RET1 was cloned by complementation and found to encode the alpha subunit of coatomer . While temperature-sensitive for growth, the newly isolated coatomer mutants exhibited a very modest defect in secretion at the nonpermissive temperature . Coatomer from beta'-COP (sec27-1) and alpha-COP (ret1-1) mutants, but not from gamma-COP (sec21) mutants, had lost the ability to bind dilysine motifs in vitro . Together, these results suggest that coatomer plays an essential role in retrograde Golgi-to-ER transport and retrieval of dilysine-tagged proteins back to the ER.

Gene, 1994 Dec 30, 151(1-2), 291 - 6
Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family; Honore B et al.; We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database {Celis et al., Electrophoresis 14 (1993) 1091-1198} . The deduced aa sequence contains 9 Trp residues, some of which are localized in repeats and that characterise the protein as a member of the WD-40 family, a group of proteins having 40-aa repeats containing Trp and Asp {Duronio et al., Proteins 13 (1992) 41-56; Van der Voorn and Ploegh, FEBS Lett . 307 (1992) 131-134} . The protein contains a nuclear targeting signal (KKKGK), and fractionation of transformed human amnion cells (AMA) in karyoplasts and cytoplasts confirmed that it is predominantly localized in the nucleus . Database searching indicated that IEF SSP 9502 is a putative human homologue of the Saccharomyces cerevisiae periodic Trp protein, PWP1, a polypeptide that may play a regulatory role in cell growth and/or transcription.

Gene, 1994 Dec 30, 151(1-2), 279 - 84
Cloning and analysis of human cDNAs encoding a 140-kDa brain guanine nucleotide-exchange factor, Cdc25GEF, which regulates the function of Ras; Wei W et al.; Ras proteins bound to GDP are biologically inactive while those bound to GTP are active . Ras-specific guanine nucleotide-exchange factors (GEFs) have been shown to activate Ras proteins . We used oligodeoxyribonucleotide primers with sequences similar to the cDNAs of rat and mouse cdc25 (encoding a Ras-GEF) to amplify, by the PCR, sequences with the potential to encode a 1275-amino-acid protein homologous to the rodent Cdc25GEF proteins . Northern blot analysis detected a brain-specific 5-kb transcript . We provide evidence for a novel alternately spliced transcript of cdc25 and show that these alternately spliced transcripts are differentially expressed in various regions of the adult nervous system . Antibodies raised against the C terminus of the protein recognize a 140-kDa protein in brain extracts of human, rat, guinea pig and cow; the 140-kDa protein is associated predominantly, if not exclusively, with a crude membrane fraction of brain . The C terminus of human Cdc25GEF can complement the loss of CDC25 function in Saccharomyces cerevisiae . A glutathione S-transferase fusion protein containing the C terminus of the cdc25 product can stimulate guanine nucleotide exchange on H-Ras in vitro . Further, the Cdc25-fusion protein binds tightly to the nucleotide-free form of H-Ras in vitro, and this binding is reversed by the addition of GTP.

J Biol Chem, 1994 Dec 30, 269(52), 32774 - 80
A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia . Purification, characterization, and cloning; Klumpp S et al.; We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C) . Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane . All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller . The NH2-terminal was blocked . Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes . The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction . The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan . Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium . A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C . Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme . Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure . We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively) . However, the amino acid sequences of the spacer regions are unrelated . In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity . A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5745 - 52
RNA editing of mat-r transcripts in maize and soybean increases similarity of the encoded protein to fungal and bryophyte group II intron maturases: evidence that mat-r encodes a functional protein; Thomson MC et al.; We present evidence that transcripts of the mat-r (maturase-related) genes of maize and soybean contain 15 and 14 uridines (U), respectively, at positions occupied by cytosines (C) in the mat-r gene sequences . Eleven and twelve of these C-->U edits result in an amino acid replacement . Ten C-->U edits are at corresponding nucleotides in the maize and soybean transcripts and, except for a single silent edit, the remainder are at positions in one species that are Us in the other species . This results in an increase in amino acid sequence similarity of the maize and soybean MAT-R proteins . Further, of those amino acids in maize and soybean MAT-R proteins specified by edited codons, ten are conserved in the reverse transcriptase-associated and RNA splicing-associated sequences of the cox1-I2 and/or the cox1-I1 maturases of the fungus Saccharomyces cerevisiae and the bryophyte, Marchantia polymorpha, respectively . The implied strong selection for amino acid sequence conservation indicates that the MAT-R protein is functional . The possibility is discussed that initiation of translation of the mat-r transcripts is at a four nucleotide codon, ATAA or ATGA.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5723 - 8
Cloning and expression of the Xenopus and mouse Msh2 DNA mismatch repair genes; Varlet I et al.; Bacterial MutS protein and its yeast and human homologs MSH2 trigger the mismatch repair process by their initial binding to mispaired and unpaired bases in DNA . We describe the cloning and sequencing of genes from Xenopus laevis and Mus musculus encoding the homolog of the Saccharomyces cerevisiae MSH2 (the major DNA mismatch binding protein) . Mutations in the human homolog of this gene have recently been implicated in microsatellite instability and DNA mismatch repair deficiency in tumour cells from patients with the most common hereditary predisposition to cancer (Lynch syndrome, or hereditary non-polyposis colorectal cancer, HNPCC), as well as in a significant percentage of sporadic tumours . Expression of the amphibian and murine Msh2 gene in different tissues appears to be ubiquitous . The Xenopus gene is highly expressed in eggs, a model system for the biochemistry of DNA mismatch repair . Expression of the murine gene is low in all tissues examined, and is relatively high in a rapidly dividing cell line . These data are suggestive of a role for MSH2 during DNA replication.

Science, 1994 Dec 23, 266(5193), 2007 - 11
ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates; Pazin MJ et al.; GAL4-VP16-mediated nucleosome reconfiguration and transcriptional activation were observed with preassembled chromatin templates that contained regular and physiological nucleosome spacing . Both processes were dependent on adenosine triphosphate (ATP), although binding of GAL4-VP16 to the chromatin was ATP-independent . Factor-mediated nucleosome reconfiguration was not, however, sufficient for transcriptional activation . These experiments recreate in vitro the active participation of nucleosomal cores in the regulation of transcription that occurs in vivo, and they suggest a multistep pathway for transcriptional activation in which factor- and ATP-dependent nucleosome reconfiguration is followed by facilitation by the DNA-bound activator of transcription from the repressed chromatin template.

J Biol Chem, 1994 Dec 23, 269(51), 32027 - 30
Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue; Stan R et al.; The yeast TOR1 and TOR2 proteins were previously discovered as putative targets of the immunosuppressive drug rapamycin . Although their cellular function is unknown, they are predicted to be at least 215 kDa in size and possess a C-terminal phosphatidylinositol (PI) kinase-related domain . We previously identified a conserved Ser residue, within the PI kinase-related domain of both yeast TOR proteins (Ser1972 in TOR1; Ser1975 in TOR2), as being the site of missense mutations conferring dominant rapamycin resistance . The Ser1972/1975 residue of yeast TOR is conserved in mammalian TOR homologs . One possibility is that this residue is critical for a direct interaction between TOR and the FKBP12-rapamycin complex . There is very recent biochemical evidence for an interaction between mammalian TOR and FKBP12-rapamycin (Brown, E . J., Albers, M . W., Shin, T . B., Ichikawa, K., Keith, C . T., Lane, W . S., and Schreiber, S . L . (1994) Nature 369, 756-758; Sabatini, D . M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S . H . (1994) Cell 78, 35-43) . Using the yeast two-hybrid system, we now have obtained genetic proof of a physical interaction between FKBP12-rapamycin and TOR and have demonstrated that this interaction requires the conserved Ser residue . We have found that a small fragment of wild-type yeast TOR2 spanning Ser1975 is capable of interacting with human FKBP12 in the presence of rapamycin, whereas an Arg1975 mutant fails to interact . This effect is dependent upon rapamycin and is antagonized by FK506.

Biochemistry, 1994 Dec 20, 33(50), 15204 - 14
Structure of the mouse dipeptidyl peptidase IV (CD26) gene; Bernard AM et al.; Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is an ectopeptidase whose expression is modulated during thymocyte differentiation and T cell activation . We describe here the organization of the mouse DPP IV gene . This gene, which encompasses more than 90 kb, is composed of 26 exons separated by introns, the lengths of which vary from 100 bp to more than 20 kb . Reverse PCR performed on RNA from different tissues indicated that DPP IV transcripts do not contain alternatively spliced CDS sequences and, therefore, are supposed to yield a single polypeptide . However, two types of specific mRNA have been detected that differ in their 3'UTR sequences . They derive from alternative polyadenylation of the DPP IV primary transcript, since the different 3'UTR sequences are contiguous in the mouse DPP IV gene . Sequence analysis of the gene 5'-flanking region revealed several structural features found in the TATAA-box-less promoters, including a G+C-rich segment, a high frequency of dinucleotide CpG, and an imperfect symmetrical dyad . The DPP IV gene was assigned by in situ hybridization to the mouse {2C2-2D} region, which is syntenic with human chromosome 2 . These data indicate that the human Dpp4 locus is located within this synteny region (i.e., 2q14-q37) . The genomic organization of the mouse DPP IV gene is compared to that of classical serine proteases and serine hydrolases . As structural and mechanistic conservation in the absence of sequence similarity is the most remarkable feature among alpha/beta hydrolases {Ollis, D . L., et al . (1992) Protein Eng . 5, 197-211}, we report the possible evolutionary link between the DPP IV related family and alpha/beta hydrolases.

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