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-acetylgluco