Gene, 1996 Oct 17, 176(1-2), 211 - 4 Isolation and characterization of a novel secretory protein, stromal cell-derived factor-2 (SDF-2) using the signal sequence trap method; Hamada T et al.; With use of the signal sequence trap method, we isolated a cDNA encoding a novel secretory protein, SDF-2, from the mouse stromal cell line, ST2 . The human homologue of SDF-2 was also isolated . The amino acid (aa) sequences deduced from both the clones were conserved more than 92% . The chromosomal localization of the human SDF-2 gene was mapped to 17q11.2 . The aa sequence of SDF-2 shows similarity to those of yeast dolichyl phosphate-D-mannose:protein mannosyltransferases, Pmt1p {Strahl-Bolsinger et al . (1993) Proc . Natl . Acad . Sci . USA 90, 8164-8168} and Pmt2p {Lussier et al . (1995) J . Biol . Chem . 270, 2770-2775}, whose activities have not been detected in higher eukaryotes. Mol Gen Genet, 1996 Oct 16, 252(5), 503 - 9 Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis; Spellig T et al.; Pathogenic development of Ustilago maydis, the causative agent of corn smut disease, is a multistep process . Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously . Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur . In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) from Aequorea victoria as a vital marker in U . maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo. Genomics, 1996 Oct 15, 37(2), 200 - 10 Analysis of three deletion breakpoints in Xp21.1 and the further localization of RP3; Brown J et al.; The gene responsible for X-linked retinitis pigmentosa (xlRP) in Xp21.1 (RP3) was initially localized by deletion analysis to within a 150- to 170-kb region between the CYBB locus and the proximal deletion junction (BBJPROX) from a patient, BB, who suffered from Duchenne muscular dystrophy (DMD), McLeod syndrome, chronic granulomatous disease (CGD), and xlRP . This gene has recently been isolated and was found to be located outside and 400 kb proximal to the BB deletion . Further analysis of BBJPROX has identified the breakpoint junction sequence, showing that it occurs within an Alu repetitive element on the proximal side but with no significant homology to the distal sequence in dystrophin intron 30 . Analysis of an overlapping deletion in patient NF, who suffered from DMD, CGD, and McLeod syndrome, shows that this deletion is within 4 kb but extends centromeric to BBJPROX, consistent with the location of RP3 outside the BB deletion region . A sequence with strong homology to a THE-1 transposon-like element was identified 7-13 kb from the proximal BB and NF breakpoints . These elements have been implicated in several highly unstable genomic regions . A third overlapping deletion, in a patient, SB, who suffered from CGD, McLeod syndrome, and xlRP, has here been shown to extend 380 kb proximal to the NF breakpoint, consistent with the finding that RP3 lies outside the BB deletion . This deletion has now been shown to disrupt the RP3 (RPGR) gene . The reason for the retinitis pigmentosa phenotype in patient BB remains unclear, but the most likely explanations include a long-range chromosomal position effect, a small secondary rearrangement, and the presence of a coincident autosomal form of retinitis pigmentosa. Nucleic Acids Res, 1996 Oct 15, 24(20), 4034 - 41 Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches; Chen CN et al.; Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate . yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced . The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses . One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches . The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA. Eur J Biochem, 1996 Oct 15, 241(2), 564 - 71 Cloning and characterization of cDNA for adenosine kinase from mammalian (Chinese hamster, mouse, human and rat) species . High frequency mutants of Chinese hamster ovary cells involve structural alterations in the gene; Singh B et al.; The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells . In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest . Using microsequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells . The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively . In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced . Transfection of an adenosine-kinase-deficient mutant (selected for resistance to the adenosine analog toyocamycin) of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell . The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild-type Chinese hamster ovary cells . The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase . In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency (10(-4)-10(-3)) . Results of Southern and northern-blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene . Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C-adenosine analogs . Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine-guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase . The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11837 - 41 Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders; Stilgenbauer S et al.; Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD) . Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma . other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis . To date, the critical genomic segment and candidate genes involved in these deletions have not been identified . In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization . As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3 . In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1) . Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment . Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster . This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD. Blood, 1996 Oct 15, 88(8), 3109 - 15 Minimal region of loss at 13q14 in B-cell chronic lymphocytic leukemia; Bullrich F et al.; Allelic loss at nonrandom chromosomal sites is thought to mark the position of tumor suppressor genes involved in the pathogenesis and progression of human malignancies . Solid tumors in particular have been found to harbor multiple genetic changes resulting in loss of function mutations . Tumor suppressor genes have also been found to be involved in the progression of lymphoid tumors . Previous reports have suggested the involvement of a tumor suppressor gene located on the long arm of chromosome 13, between the retinoblastoma (RB) and D13S25 loci, in the pathogenesis and or progression of more than 40% of B-cell chronic lymphocytic leukemia (B-CLL), a common lymphoid malignancy whose molecular etiology remains largely unknown . In the present study, we report the construction and characterization of a YAC contig spanning a region of approximately 3 cM between the RB gene and the D13S31 locus . We also screened 60 paired normal/tumor B-CLL samples for allelic loss on chromosome 13 with nine microsatellite markers located between RB and D13S25 . This analysis has allowed us to narrow the smallest region of loss to a segment of 550 kb located between the 206XF12 and D13S25 markers. FEMS Microbiol Lett, 1996 Oct 15, 144(1), 67 - 71 Utilization of acetonitrile and other aliphatic nitriles by a Candida famata strain; Linardi VR et al.; A variant of a yeast strain identified as Candida famata isolated from gold mine effluent was able to grow on acetonitrile, acrylonitrile, butyronitrile, isobutyronitrile, methacrylnitrile, propionitrile, succinonitrile, valeronitrile, acetamide, isobutyamide, and succinamide as sole nitrogen source, after acclimatization . The yeast grew on acetonitrile and acetamide at concentrations up to 4% . The utilisation of acetonitrile and acetamide by the C . famata strain probably involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase. Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 311 - 7 Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase; Ullah AH et al.; The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant . Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0 . Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5 . Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme . Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines . Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity. Science, 1996 Oct 11, 274(5285), 242 - 6 Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores; Chen RH et al.; The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle . The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized . A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts . XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores . This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors. Gene, 1996 Oct 10, 175(1-2), 233 - 40 Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain; Nicolas RH et al.; A number of transcription factors that act as adaptor proteins have been found to contain an 87 amino acid domain called the bromodomain . In a study to identify and characterise bromodomain proteins expressed in chicken cells, a novel gene has been isolated which encodes five repeats of the bromodomain . In addition, the encoded protein, termed polybromo, contains four other domains: an unusual truncated HMG box, two repeats of a novel domain which we term the BAH domain and a sequence related to a region within the regulatory domain of the DNA cytosine-5 methyltransferase enzyme . Polybromo was found to be related to a yeast protein U19102 which has two bromo domains, a BAH domain and the DNA methyltransferase-related sequence . Antibodies that were raised against polybromo were used in confocal microscopy analysis to show that the 180-kDa polybromo protein is located within the nucleus but excluded from the nucleolus . Gel filtration analysis of nuclear extracts demonstrate that polybromo is part of a large complex with a mass of approximately 2 million dalton. Biochemistry, 1996 Oct 8, 35(40), 13157 - 64 ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis; Zeng C et al.; The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable . It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase . On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase . Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP . Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu . The kcat values decreased 1000- and 200-fold, respectively, for the two mutants . Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser . The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group . The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme . The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants . The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme . The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state. J Mol Biol, 1996 Oct 4, 262(4), 559 - 74 Protein globularization during folding . A study by synchrotron small-angle X-ray scattering; Semisotnov GV et al.; Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS) . SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots . Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass . The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra . The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters . It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration . We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions . Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding . Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded . The kinetic processes reflect both protein intramolecular globularization and its intermolecular association. Cell, 1996 Oct 4, 87(1), 65 - 73 Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis; Umar A et al.; A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins . PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2 . A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1 . Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis . Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA . The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis . The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions. J Biol Chem, 1996 Oct 4, 271(40), 24811 - 6 Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9; Wang ZY et al.; We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays . The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes . The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9 . Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested . A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3 . We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system. J Biol Chem, 1996 Oct 4, 271(40), 24675 - 83 Distinct domains of myocyte enhancer binding factor-2A determining nuclear localization and cell type-specific transcriptional activity; Yu YT; The myocyte enhancer binding factor 2 (MEF2) family of transcription factors plays an important role in the regulation of gene expression in multiple muscle cell types . Four mef2 genes (A, B, C, and D) have been identified in vertebrates . They share the conserved N-terminal MCM1-agamous-deficiens-serum response factor and MEF2 domains that determine DNA binding and dimerization functions . In this study, we have identified human MEF2A domains that control its nuclear localization and transcriptional activation function . Studies of subcellular localization of various truncated MEF2A proteins expressed in HeLa cells demonstrated that MEF2A contains a nuclear localization signal located at its C terminus within the peptide encompassing amino acids (aa) 472-507 . Examination of MEF2A mutants with sequential C-terminal deletions indicates that the region encompassing aa 321-472 is essential for transcriptional activity . Analysis of the transcriptional activity of fusion proteins consisting of different domains of MEF2A fused to the DNA binding domain of yeast transcription factor GAL4 revealed a major transcriptional activation domain located between aa 274 and 373 . Further studies demonstrated that this domain contains positive and negative regulatory subdomains that cooperate with one another to regulate the transcriptional activity of MEF2A in a cell-type discriminatory manner. J Biol Chem, 1996 Oct 4, 271(40), 24583 - 9 c-Jun can mediate androgen receptor-induced transactivation; Bubulya A et al.; The proto-oncoprotein c-Jun forms as a heterodimer with c-Fos, the transcription factor AP-1 . AP-1 regulates transcription through transactivation, a process requiring DNA binding . Here we report an indirect mechanism by which c-Jun can regulate transcription via the androgen receptor . In this process, c-Jun is able to support androgen receptor-mediated transactivation in the absence of an interaction with c-Fos or any apparent DNA binding . This positive effect of c-Jun was dose-dependent . Both exogenously added and endogenously induced c-Jun are able to act on the androgen receptor . Transactivation by the androgen receptor can undergo self-squelching, and this was relieved by transfected c-Jun . Using a time-course experiment, we provide evidence that the c-Jun effect is primary . c-Fos is able to block human androgen receptor activity in both the absence and presence of transfected c-Jun . Using a modified form of the yeast two-hybrid system, we show in Cos cells that c-Jun can interact with the DNA binding domain/hinge region (CD regions) of the androgen receptor . Therefore, we propose that c-Jun functions as a mediator for androgen receptor-induced transactivation. J Biol Chem, 1996 Oct 4, 271(40), 24564 - 8 Role of hydrophobic amino acids at beta85 and beta88 in stabilizing F helix conformation of hemoglobin S; Reddy LR et al.; Three Hb S variants containing Glu substitutions at Phe-beta85 and/or Leu-beta88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S . Helix stability of tetrameric Hb S betaF85E,betaL88E was measured by CD and compared with those of Hb S betaF85E, Hb S betaL88E, Hb A, and Hb S . The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10 degrees C . However, changes in ellipticity at 222 nm for Hb S betaF85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S betaL88E and Hb S betaF85E,betaL88E were similar and about 30-fold greater than Hb S . Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S betaF85E,betaL88E was similar to that of Hb S betaL88E rather than Hb S betaF85E . These results suggest that Glu insertion at both beta85 and beta88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S betaF85E, betaL88E is similar to Hb S betaL88E rather than Hb S betaF85E . Furthermore, these results suggest that both Phe-beta85 and Leu-beta88 are critical for F helix stabilization and that Glu insertion at beta88 leads to more destabilization than insertion at beta85. J Biol Chem, 1996 Oct 4, 271(40), 24557 - 63 Polymerization of three hemoglobin A2 variants containing Val6 and inhibition of hemoglobin S polymerization by hemoglobin A2; Adachi K et al.; To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valdelta6 Hb A2 variants (Hb A2 deltaE6V, Hb A2 deltaE6V,deltaQ87T, and Hb A2 deltaE6V, deltaA22E,deltaQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed . Oxy forms of Hb A2 deltaE6V and Hb A2 deltaE6V,deltaQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 deltaE6V,deltaA22E,deltaQ87T was similar to that of Hb S, suggesting that Aladelta22 and Glndelta87 contribute to the surface hydrophobicity of Hb A2 . Deoxy Hb A2 deltaE6V polymerized without a delay time, like deoxy Hb F gammaE6V, while deoxy Hb A2 deltaE6V,deltaQ87T and deoxy Hb A2 deltaE6V,deltaA22E,deltaQ87T polymerized after a delay time, like deoxy Hb S, suggesting that beta87 Thr is required for the formation of nuclei . Deoxy Hb F gammaE6V,gammaQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valdelta6 Hb A2 and Valgamma6 Hb F variants are different . Mixtures of deoxy Hb S/Hb A2 deltaE6V,deltaQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 deltaE6V mixtures was inhibited, like Hb S/Hb F gammaE6V mixtures . These results suggest alpha2betaSdelta6 Val, 87 Thr hybrids and Hb A2 deltaE6V,deltaQ87T participate in Hb S nucleation, while only 50% of alpha2betaSdelta6 Val hybrids and none of the Hb A2 deltaE6V participate . These findings are in contrast to those of mixtures of Hb S with Hb F gammaE6V or Hb F gammaE6V,Q87T, which both inhibit Hb S polymerization . Our results also suggest participation in nucleation of some alpha2betaSdelta hybrids in A2S mixtures but not alpha2betaSgamma hybrids in FS mixtures. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 152 - 9 Evidence that the human homologue of a rat initiation factor-2 associated protein (p67) is a methionine aminopeptidase; Li X et al.; Previously, we cloned a human cDNA encoding a protein which has a 92% amino acid sequence identity to a rat initiation factor-2 associated protein (p67) . Rat p67 plays an important role in translational regulation by preventing the phosphorylation of the alpha subunit of initiation factor-2 . Interestingly, several lines of indirect evidence suggested that this protein may also function as a methionine aminopeptidase (MetAP) . To test this hypothesis, we expressed the human cDNA in a baculovirus system, purified it to homogeneity and characterized it . Using 13 different peptide substrates, we found that the human p67 has a similar substrate specificity with other MetAPs . Kinetic analyses revealed that the Kcat/K(m) values of the human MetAP on two representative substrates are similar to those of yeast and porcine MetAPs . Furthermore, we found that this enzyme, like other MetAPs, is also a cobalt-dependent metalloenzyme. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 82 - 7 The metastasis suppressor candidate nucleotide diphosphate kinase NM23 specifically interacts with members of the ROR/RZR nuclear orphan receptor subfamily; Paravicini G et al.; We have cloned proteins that interact with the nuclear orphan receptor RZR beta using the yeast two-hybrid system . We identified, amongst a number of other genes, the nucleoside diphosphate kinase (NDPK)-2 also known as Nm23-2, c-myc regulatory factor PuF and differentiation inhibitory factor, RZR beta specifically interacts with Nm23-2 but not with the closely related tumor metastasis suppressor candidate gene product Nm23-1 . In contrast ROR alpha interacts with both Nm23 proteins . These findings were corroborated by in vitro interaction assays based on GST-pulldown experiments . With-n-myc we propose a candidate gene regulated by ROR alpha/RZR beta and Nm23, based on the finding that the respective DNA binding sites in the first intron are conserved in several mammalian species. Nature, 1996 Oct 3, 383(6599), 453 - 7 A new group of conserved coactivators that increase the specificity of AP-1 transcription factors; Claret FX et al.; The Jun proteins are nuclear proteins that combine with Fos proteins to form a gene-regulatory protein, AP-1 . They have highly conserved DNA-binding and dimerization domains, resulting in almost identical sequence-recognition properties . Nevertheless, there are many indications that each Jun protein activates a distinct and only partially overlapping set of AP-1 target genes . Using the more variable activation domain of c-Jun as a bait, we identified a protein, JAB1, that interacts with c-Jun and JunD, but not with JunB or v-Jun . As a result, JAB1 selectively potentiates transactivation by only c-Jun or JunD . In vitro, JAB1 specifically stabilizes complexes of c-Jun or JunD with AP-1 sites and does not affect binding of either JunB or v-Jun . The amino-terminal half of JAB1 is very similar to the amino terminal region of Pad1 from fission yeast, which was identified genetically as a coactivator of a subset of AP-1 target genes . JAB1 and Pad1 are also functionally interchangeable . They define a new group of coactivators that increase the specificity of target gene activation by AP-1 proteins. Biochim Biophys Acta, 1996 Oct 2, 1284(1), 9 - 12 Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein; Iacobazzi V et al.; The tricarboxylate or citrate transporter protein (CTP) catalyzes the transport of citrate across the inner mitochondrial membrane by an exchange for malate or some other anionic metabolite . Using primers based on the rat liver cDNA sequence, overlapping cDNA clones encoding the bovine CTP were isolated from bovine liver poly(A+) cDNA . The entire bovine cDNA is 1151 bp in length with 5' and 3' untranslated regions of 7 and 204 bp, respectively . The open reading frame encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids . The amino acid sequence of the mature bovine CTP is 95.6, 94.9, 32.2% identical to that of the citrate carrier from man, rat and yeast, respectively. Cell Struct Funct, 1996 Oct, 21(5), 421 - 4 A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli; Yahara I et al.; 1 . Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes . The structure and function of cofilin are conserved during evolution . 2 . Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3 . Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo . 3 . The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells . 4 . In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue . The actin-binding activity is essential for yeast cells to grow . 5 . Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells . 6 . Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses . Functional roles of cofilin/actin in the nucleus remain to be elucidated . 7 . Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity. Semin Cancer Biol, 1996 Oct, 7(5), 299 - 306 Complementation tagging of cooperating oncogenes in knockout mice; Allen JD et al.; Gene disruption and overexpression in mice can be combined with proviral tagging to investigate the biochemical pathways in which oncogenes act . The phenomenon of oncogene collaboration permits a focussed genetic approach analogous to the strategies employed in suppressor screens in invertebrates and yeast . We illustrate here its application to investigating the action of the pim kinases in tumorigenesis . However, it should be possible to utilize this strategy for dissecting a much wider range of biochemical pathways in mammalian systems. Bioorg Khim, 1996 Oct-Nov, 22(10-11), 870 - 4 {Precipitation of cytochromes c with complex cadmium iodide}; Zhuravleva DV et al.; Using cytochrome c from horse heart and the yeast candida valida as examples, it was shown that a complex anion, cadmium tetraiodide (CdI42-), precipitated proteins from aqueous solutions at the reagent concentrations below 50 mM . The composition and pH value of the solution, as well as the starting protein concentration, considerably influenced the precipitation . The results suggest that this reagent acts on the protein by a mechanism similar to the salting-out process . The ability to act at small concentrations is the advantage of CdI42- over conventional agents. Med Parazitol (Mosk), 1996 Oct-Dec, (4), 43 - 5 {A dry nutrient medium for the cultivation of Leishmania promastigotes}; Osokina TI et al.; A dry medium formulation which provides the growth and reproduction of Leishmania promastigotes was designed . The medium has been prepared by using nondietary protein raw material, it contains domestic-production ingredients of that in high supply . The nutrient basis of the medium is enzymatic peptone (a source of amine nitrogen) . The growth factors of Leishmania (a yeast extract, vitamins, amino acids, a dry blood derivative), carbohydrates (an energy components of the medium), inorganic salts (a source of trace elements), which are selected in strictly defined quantitative ratios, meet the physiological requirements of Leishmania . The agent rules out the necessity of adding ex tempore blood, making the process for preparing the medium from the dry powder simple and easy-to-use. Clin Genet, 1996 Oct, 50(4), 267 - 9 FISH characterization of the Xq21 breakpoint in a translocation carrier with premature ovarian failure; Riva P et al.; A panel of ordered YAC clones, isolated using STSs in the Xq13-Xq23 region, was used to characterize by Fluorescent In Situ Hybridization (FISH) the Xq21 breakpoint in a t(X;1)(q21;p34) translocation female with premature ovarian failure . The YAC 949E11 was found to span the breakpoint, but also to join the two non-overlapping YACs 36CB1 and 40AB3, proximal and distal, respectively, to the patient's Xq21 breakpoint. Lett Appl Microbiol, 1996 Oct, 23(4), 253 - 6 Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803; Onishi N et al.; Galacto-oligosaccharide (Gal-OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803 . With toluene-treated resting cells, 136 mg ml-1 of Gal-OS was produced from 360 mg ml-1 of lactose at 50 degrees C for 42 h . Then, the yield of Gal-OS was increased by a culture method in which cell growth followed the enzymatic reaction: 224 mg ml-1 of Gal-OS was produced at 30 degrees C for 60 h . Finally, combination of the toluene-treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by-product, glucose, which inhibits the Gal-OS production, from the reaction mixture . In this case, 242 mg ml-1 of Gal-OS was produced at 50 degrees C for 42 h without cell growth . The structure of the major product was identified as 4'-galactosyl-lactose. Plant Mol Biol, 1996 Oct, 32(1-2), 1 - 41 Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery; Simpson GG et al.; The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing . It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated . The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome . The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here . Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals . A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing . In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing . We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing . Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei . Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago . The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains. Genet Anal, 1996 Oct, 13(4), 99 - 103 Identification and mapping of a putative bombesin receptor gene on human chromosome 17q21.3+; Kelavkar U et al.; A mouse bombesin receptor cDNA was used as a probe to screen a human P1 genomic library . Clone HBR1 was isolated and used to localize a putative human bombesin receptor gene (HBRKS) on human chromosome 17q21.3 by fluorescent in situ hybridization (FISH) . HBRKS was identified and mapped by polymerase chain reaction (PCR) amplification from a Yeast artificial chromosome (YAC) contig spanning 17q21-q23 . In addition, a few candidate genes were found by exon-trapping from HBR1. J Med Genet, 1996 Oct, 33(10), 842 - 7 Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome; Drumheller T et al.; In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation . We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?) . EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses . The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25 . The analysis clearly shows that all five breakpoints are distinct . Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients . Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease . The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints . This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development. Environ Health Perspect, 1996 Oct, 104(10), 1084 - 9 Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays; Klotz DM et al.; Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans . To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity . Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES) . This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast . Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay . Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene . Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity . Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models . Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays . The antifungal agent benomyl had no estrogenic activity . We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity. Mol Cell Biol, 1996 Oct, 16(10), 5865 - 75 Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis; Ema M et al.; From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim) . Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt . In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex . This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z . Chang, D . Price, S . Bockheim, M . J . Boedigheimer, R . Smith, and A . Laughon, Dev . Biol . 160:315-322, 1993; D . M . Mellerick and M . Nirenberg, Dev . Biol . 171:306-316, 1995) . Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2) . From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome . The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome. Acta Paediatr, 1996 Oct, 85(10), 1143 - 5 Selenium supply and glutathione peroxidase activity in breastfed Polish infants; Trafikowska U et al.; Selenium (Se) concentration in human milk in Poland is below 10 ng ml-1 and the Se intake by breastfed infants is about 6 micrograms day-1 . Supplementation of lactating mothers with selenium-enriched yeast increases rapidly and significantly the Se concentration and glutathione peroxidase activity in maternal blood components . Se concentration in milk is also significantly elevated . After 1 month the mean Se intakes by breastfed infants were greater than the recommended dietary allowance of 10 micrograms day-1 for infants from birth to 6 months of age. Anticancer Drug Des, 1996 Oct, 11(7), 493 - 508 Biochemical and chemical characterization of phenylglyoxal bis(guanylhydrazone), an aromatic analogue of mitoguazone; Elo H et al.; Since little has been known about the properties of aromatic analogues of the antineoplastic agent methylglyoxal bis(guanylhydrazone) (MGBG), an investigation was performed on phenylglyoxal bis(guanylhydrazone) (PhGBG) . PhGBG competitively inhibited yeast adenosylmethionine decarboxylase (AdoMetDC) with a Ki of 65 microM . As compared to MGBG (Ki 0.23 microM), PhGBG is a much weaker inhibitor, being even weaker than the unsubstituted congener glyoxal bis(guanylhydrazone) (GBG, Ki 18 microM) . PhGBG inhibited porcine kidney diamine oxidase (DAO) non-competitively, being a more potent inhibitor (Ki 0.12 microM) than GBG (Ki 0.17 microM) or MGBG (Ki 0.33 microM) . Thus, PhGBG has an unfavourably high ratio of Ki(AdoMetDC)/Ki(DAO) for potential use for selectively inhibiting polyamine biosynthesis . This does not exclude the possibility that PhGBG or other aromatic congeners might have therapeutic value since the corresponding ratio of the antileukaemic congeners GBG and MGBG is also high as compared to many aliphatic non-antileukaemic analogues . The pKa1 and pKa2 values of PhGBG dication were found to be 6.39 +/- 0.02 and 8.64 +/- 0.02 respectively, their difference being distinctly larger than in the case of GBG or its C-alkylated analogues . This may result from decreased stability of the dication form, caused by the resonance effect or possibly by the inductive effect of the phenyl group . The species distribution of PhGBG (proportion of free base 5.5%, predominant species the monocation) at 37 degrees C resembles that of GBG and MGBG but is clearly different from that of non-antileukaemic C-alkylated analogues . These similarities suggest that PhGBG and its derivatives may be worth antitumour screening . Depending on the conditions used in the crystallization, three different types of crystals of PhGBG sulphate were obtained . Crystallography indicated that, in two of the types, the crystal consisted exclusively of the anti-anti isomer, i.e . the same isomer as has been observed in the case of GBG and its C-alkylated congeners . One crystal type, however, consisted of a different geometrical isomer (anti-syn), suggesting that PhGBG may isomerize more easily than its aliphatic analogues . Previous concepts on the isomerism of GBG and C-alkylated bis(guanylhydrazones) thus cannot be generalized to aromatic congeners . A theory based on resonance, inductive and hyperconjugative effects and electron transfers is presented that is capable of explaining the formation of the two geometrical isomers of PhGBG that were experimentally observed . A similar theory, based on hyperconjugation of C-F bonds, is presented that is capable of explaining the previous finding of the formation of the anti-syn isomer of trifluoromethylglyoxal bis(guanylhydrazone) (CF3GBG) . Like that of CF3GBG, the anti-syn isomer of the PhGBG dication is stabilized by an internal hydrogen bond . The lack of structural rigidity may affect the biological properties of PhGBG, e.g . its ability to inhibit AdoMetDC. Genomics, 1996 Oct 1, 37(1), 24 - 8 The B-lymphocyte maturation promoting transcription factor BLIMP1/PRDI-BF1 maps to D6S447 on human chromosome 6q21-q22.1 and the syntenic region of mouse chromosome 10; Mock BA et al.; The human PRDI-BF1 or BLIMP1 gene and its mouse homolog Blimp1 are members of the recently realized PR domain family that includes the retinoblastoma interacting zinc finger gene RIZ and the MDS1-EVI1 leukemia cancer gene . The specific high-level expression of Blimp1 in late B and plasma cells, its induction during B-cell differentiation, and its ability to drive B-cell maturation suggest that this gene may play a role in the differentiation and pathogenesis of B cells . We have now mapped the physical location of BLIMP1 near the marker D6S447 on human chromosome 6q21-q22.1; we have also mapped Blimp1 to mouse Chromosome 10 at 14 cM distal to the Myb locus and to a region homologous to the location of BLIMP1 . Deletions of the 6q21-q22 region are common in several human malignancies, particularly in B-cell non-Hodgkin lymphoma (B-NHL) . The data led us to suggest that BLIMP1 may be a candidate B-NHL tumor suppressor gene. Genomics, 1996 Oct 1, 37(1), 9 - 18 High-density radiation hybrid map of human chromosome 18 and contig of 18p; Giacalone J et al.; A high-resolution radiation hybrid map of human chromosome 18 has been developed by testing DNA samples of 92 radiation hybrids (RH) from a previously characterized chromosome 18-specific RH panel . Half of the 159 STS markers were tested on RH DNA amplified by primer extension preamplification . This map includes 20 genes and 95 polymorphic markers, most of which have heterozygosity frequencies greater than 65% and, therefore, allow integration with genetic maps . The framework map consists of 137 markers ordered with odds of 1000:1 or better and spaced on average 580 kb apart . A YAC contig map of 18p was generated independently by STS content mapping of YACs using the same primers as for RH mapping . The RH map and the contig map are concordant. Genes Chromosomes Cancer, 1996 Oct, 17(2), 108 - 17 Fluorescence in situ hybridization deletion mapping at 4p16.3 in bladder cancer cell lines refines the localisation of the critical interval to 30 kb; Bell SM et al.; An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours . In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus . To refine this region of deletion at 4p16.3, we have carried out detailed fluorescence in situ hybridisation (FISH) analysis of 12 bladder cancer cell lines by using a chromosome 4 centromeric probe combined with a series of cosmid probes from contigs spanning the 750 kb region of deletion . A common 30 kb region of deletion was identified at 4p16.3 in over one-third of the bladder cancer cell lines analysed . The present study has refined the localisation of the critical region of deletion from 750 kb to approximately 30 kb, providing a precise starting point for positional cloning of the gene(s) involved in bladder cancer from within a very gene-rich region on chromosome band 4p16.3 . This study demonstrates that FISH can be used for fine deletion mapping of potential tumour suppressor gene regions . The utilisation of FISH analysis to map chromosomal deletions should facilitate positional cloning of other genes as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) contigs of the human genome are established. Genes Chromosomes Cancer, 1996 Oct, 17(2), 78 - 87 Detection of chromosomal DNA gains and losses in testicular germ cell tumors by comparative genomic hybridization; Korn WM et al.; To extend the results of conventional cytogenetic analysis of testicular germ cell tumors (TGCTs), we applied the new molecular cytogenetic method of comparative genomic hybridization (CGH), which enables the detection of chromosomal imbalances without the need for dividing cells . DNA from II TGCTs was studied by CGH . In all tumors examined, gain of 12p, mostly of the whole p arm, could be demonstrated . However, in three tumors, an amplification of 12p material restricted to the chromosomal bands 12p11.2-p12.1 was found . Further fluorescence in situ hybridization (FISH) analysis using a yeast artificial chromosome (YAC) that was previously mapped to that region revealed multiple copies of that chromosomal segment in interphase nuclei of these tumors . This finding is an important clue to the localization of candidate protooncogenes at 12p involved in TGCTs . Gains of small chromosomal regions at 2p, 4q, 6p, and 19p were also detected recurrently . Furthermore, gains of chromosomes 8, 14, 21, and X as well as loss of chromosome 13 were frequent findings . In conclusion, CGH provides new insights into genetic alterations of TGCTs . By using CGH, chromosomal subregions could be identified that may harbor genes involved in the pathogenesis of this malignancy. Immunol Cell Biol, 1996 Oct, 74(5), 457 - 64 Breast cancer immunotherapy: current status and future prospects; Apostolopoulos V et al.; The development of an immunotherapeutic approach to cancer is the concern for many immunologists, but despite the impressive progress over the past decade, such as the identification of tumour antigens and antigenic peptides as potential targets, there are still many obstacles in eliciting an effective immune response to eradicate cancer . Mucins have attracted interest as potential targets for immunotherapy in the development of vaccines for cancers expressing Mucin1 (MUC1; e.g . breast, pancreas, ovary etc.) . All of the identified targets for cancer, including MUC1, are normal proteins; however MUC1 expressed on tumours can be considered as tumour specific due to their overexpression, altered glycosylation and its ubiquitous distribution on the cell surface rather than at the secretory pole in adenocarcinomas . These observations have led to the development of several different approaches to immunize against breast cancer using synthetic carbohydrates or peptides conjugated to carriers and given together with a variety of adjuvants to elicit the appropriate immune response . Mannan, a polymannose carbohydrate isolated from the cell wall of yeast, is an appropriate and effective protein carrier for eliciting a cellular (T1-type) or humoral (T2-type) immune response depending on the mode of conjugation (oxidized or reduced) . In addition, mannan holds promise and opens many avenues as a carrier for vaccine development for other antigens . Several clinical trials are in progress to evaluate the immunogenicity of MUC1 and its suitability as to use for immunotherapy/vaccine for breast cancer. Genome Res, 1996 Oct, 6(10), 1002 - 12 Utilization of FISH in positional cloning: an example on 13q22; Laan M et al.; In positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region . The construction of a high-resolution physical map still involves considerable effort . However, new high-resolution fluorescence in situ hybridization (FISH) techniques have facilitated this process substantially . Here we summarize a strategy that combines a spectrum of FISH techniques {metaphase, interphase, mechanically stretched chromosomes (MSCs), and fiber-FISH on free chromatin} for the construction and characterization of a high-resolution physical map for a positional cloning project . The chromosomal region 13q22, containing the locus of the variant form of the neuronal ceroid lipofuscinosis (vLINCL, CLN5) disease, serves here as an example for this process . We used metaphase FISH to exclude positionally a candidate gene, to refine the locus to 13q22, and to analyze the possible chimerism of the YACs in the region . Both metaphase and interphase FISH techniques were applied to determine the low-resolution distances between the restricting markers . FISH using MSCs confirmed the centromeric-telomeric order of the clones and facilitated the estimation of the size of the gaps between the clones . Finally, fiber-FISH was found to be the method of choice for the construction of an accurate high-resolution map of the contig established over the restricted region . Thus, FISH techniques in combination with genetic mapping data enabled the refinement of the initial 4-cM region to a high-resolution map of only 400 kb in length . Here the FISH strategy replaced the need for many laborious traditional physical mapping methods, e.g., pulsed-field gel electrophoresis. Genome Res, 1996 Oct, 6(10), 943 - 55 An integrated YAC map of the human X chromosome; Roest Crollius H et al.; The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males . With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map . We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones . Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources . We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones . These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques . This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps. Biochem Mol Biol Int, 1996 Oct, 40(3), 611 - 6 Primary structures and sequence analysis of human ribosomal proteins L39 and S27; Tsui SK et al.; We report here the primary structures and sequence analysis of the human ribosomal protein L39 (hRPL39) and S27 (hRPS27) . The hRPL39 cDNA is 352 bp in size encoding a predicted protein of 51 amino acids . The region KRRHWRRTKL near the carboxyl end is conserved between human, rat, maize, C . elegans and yeast . The hRPS27 cDNA is 321 bp in size encoding a deduced protein of 84 amino acids . When the deduced amino acid sequence of hRPS27 was compared with that of rat ribosomal protein S27 and human metalloproteinstimulin-1 (MPS-1), identity levels of 96.4% and 100% were obtained respectively . A potential polyadenylation signal AACAAA is found in the MPS-1 cDNA but the more frequently used AATAAA sequence is present in the hRPS27 cDNA . The carboxyl-terminal cysteine arrangement in hRPS27 is similar to the family of C4 zinc finger DNA-binding proteins. Cancer Genet Cytogenet, 1996 Oct 1, 91(1), 1 - 7 Amplification of a rearranged form of the high-mobility group protein gene HMGIC in OsA-CI osteosarcoma cells; Kools PF et al.; Recently the high-mobility group protein gene HMGIC has been found to be rearranged in a variety of benign mesenchymal tumors with 12q13-q15 aberrations, such as angiomyxoma, fibroadenomas of the breast, lipomas, pleomorphic salivary gland adenomas, polyps of the endometrium, pulmonary chondroid hamartomas, and uterine leiomyomas . Here we report on HMGIC aberrations in the osteosarcoma cell line OsA-CI . In Northern blot studies, aberrant HMGIC transcripts were detected . Analysis of cDNA sequence data obtained after 3' rapid amplification of cDNA ends, indicated these to consist of 5' HMGIC sequences encoding the three DNA binding domains fused to ectopic sequences apparently derived from part of the human lumican (keratan sulphate proteoglycan) gene (LUM), which we mapped by fluorescence in situ hybridization (FISH) to chromosome 12q22-q23 . Moreover, Southern blot analysis revealed amplification of this fusion gene but not of the 3'HMGIC sequences . This observation was independently confirmed by FISH analysis using yeast artificial chromosome (YAC) and cosmid clones, which furthermore indicated that the amplified 5'HMGIC sequences were contained within an amplicon of about 200 kb . Our results indicate that aberrations in HMGIC might not be restricted to benign mesenchymal tumors. FASEB J, 1996 Oct, 10(12), 1378 - 87 The eIF-2alpha kinases and the control of protein synthesis; de Haro C et al.; Protein synthesis is regulated in response to environmental stimuli by covalent modification, primarily phosphorylation, of components of the translational machinery . Phosphorylation of the alpha subunit of eIF-2 is one of the best-characterized mechanisms for down-regulating protein synthesis in higher eukaryotes in response to various stress conditions . Three distinct protein kinases regulate protein synthesis in eukaryotic cells by phosphorylating the alpha subunit of eIF-2 at serine-51 . There are two mammalian eIF-2alpha kinases: the double-stranded RNA-dependent kinase (PKR) and heme-regulated inhibitor kinase (HRI), and the yeast GCN2 . The regulatory mechanisms and the molecular sizes of these eIF-2alpha kinases are different . The expression of PKR is induced by interferon, and the kinase activity is stimulated by low concentrations of double-stranded RNA . HRI is activated under heme-deficient conditions . Yeast GCN2 is activated by amino acid starvation . The phosphorylation of eIF-2alpha results in the shutdown of protein synthesis . Nevertheless, the eIF-2alpha kinases can regulate both global as well as specific mRNA translation . Inhibition of protein synthesis correlates with eIF-2alpha phosphorylation in response to a wide variety of different stimuli, including heat shock, serum deprivation, glucose starvation, amino acid starvation, exposure to heavy metal ions, and viral infection . Finally, recent studies suggest a role for eIF-2alpha phosphorylation in the control of cell growth and differentiation. Development, 1996 Oct, 122(10), 2965 - 76 Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis; Banuett F et al.; Ustilago maydis is a dimorphic fungus with a yeast-like non-pathogenic form and a filamentous (hyphal) pathogenic form that induces tumor formation in maize . Within mature tumors, hyphae give rise to teliospores, which are round, diploid cells surrounded by a specialized cell wall . Here we describe the time course of fungal development in the plant with a focus on the morphological changes in the hyphae and the pathway of teliospore formation . We confirm and extend earlier observations that U . maydis hyphae branch extensively on the leaf surface and intracellularly before induction of tumors . We observe that at later stages the filaments undergo a series of discrete morphogenetic changes leading to teliospore formation . In particular, we show that the hyphae become embedded in a mucilaginous matrix within the tumor cells and the hyphal tips become modified . The hyphae then undergo fragmentation to release individual cells that exhibit a variety of shapes on their way to becoming rounded . Finally, a specialized cell wall is deposited . Support for the existence of such a pathway comes from analysis of a mutant defective in the fuz1 gene: inactivation of fuz1 blocks production of the mucilaginous matrix and fragmentation of the hyphae, leading to a defect in teliospore formation . The different morphological changes that occur while in the plant but not in culture suggest that plant inputs play a key role in fungal development. EMBO J, 1996 Oct 1, 15(19), 5370 - 82 Purification and biochemical heterogeneity of the mammalian SWI-SNF complex; Wang W et al.; We have purified distinct complexes of nine to 12 proteins {referred to as BRG1-associated factors (BAFs)} from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1 . Microsequencing revealed that the 47 kDa BAF is identical to INI1 . Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene . A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex . Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex . Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition . The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes . Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type. EMBO J, 1996 Oct 1, 15(19), 5256 - 67 Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002; Brunn GJ et al.; The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR) . The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases . This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes . This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation . A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation . In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells . At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR . The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer . These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Hum Mol Genet, 1996 Oct, 5(10), 1649 - 55 Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database; Allikmets R et al.; As an approach to characterizing all human ATP-binding cassette (ABC) superfamily genes, a search of the human expressed sequence tag (EST) database was performed using sequences from known ABC genes . A total of 105 clones, containing sequences of potential ABC genes, were identified, representing 21 distinct genes . This brings the total number of characterized human ABC genes from 12 to 33 . The new ABC genes were mapped by PCR on somatic cell and radiation hybrid panels and yeast artificial chromosomes (YACs) . The genes are located on human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17 and X; at locations distinct from previously mapped members of the superfamily . The characterized genes display extensive diversity in sequence and expression pattern and this information was utilized to determine potential structural, functional and evolutionary relationships to previously characterized members of the ABC superfamily. Hum Mol Genet, 1996 Oct, 5(10), 1589 - 98 cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein; Bressler SL et al.; Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP) . A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region . The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein . This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2 . These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP . Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID) . Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins . RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology . The human Fe65 mRNA was cloned from a fetal brain cDNA library . The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein . The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization. Hum Mol Genet, 1996 Oct, 5(10), 1547 - 57 Positional cloning of a gene involved in hereditary multiple exostoses; Wuyts W et al.; Hereditary multiple exostosis (EXT) is an autosomal dominant condition mainly characterized by the presence of multiple exostoses on the long bones . These exostoses are benign cartilaginous tumors (enchondromata) . Three different EXT loci on chromosomes 8q (EXT1), 11p (EXT2) and 19p (EXT3) have been reported, and recently the EXT1 gene was identified by positional cloning . To isolate the EXT2 gene, we constructed a contig of yeast artificial chromosomes (YAC) and P1 clones covering the complete EXT2 candidate region on chromosome 11p11-p12 . One of the transcribed sequences isolated from this region corresponds to a novel gene with homology to the EXT1 gene, and harbours inactivating mutations in different patients with hereditary multiple exostoses . This indicates that this gene is the EXT2 gene . EXT2 has an open reading frame encoding 718 amino acids with an overall homology of 30.9% with EXT1, suggesting that a family of related genes might be responsible for the development of EXT. Plant J, 1996 Oct, 10(4), 703 - 11 U-rich tracts enhance 3' splice site recognition in plant nuclei; Baynton CE et al.; The process of 5' and 3' splice site definition in plant pre-mRNA splicing differs from that in mammals and yeast . In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5' splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3' splice site; in plants, the 3' intron boundary is defined in a position-dependent manner relative to AU-rich elements within the intron . To determine if uridines are utilized to any extent in plant 3' splice site recognition, uridines in the region preceding the normal (-1) 3' splice site of pea rbcS3A intron 1 were replaced with adenosines . This mutant activates two cryptic 3' splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (-1) site are essential for recognition . Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3' splice site containing 14 or 10 uridine tracts with a G at -4 can effectively outcompete the normal 3' splice site containing an eight uridine tract with a U at -4 . Substitutions at the -4 position demonstrated that the identity of the nucleotide at this position greatly affects 3' splice site selection . It has been concluded that several factors affect competition between these 3' splice sites . These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3' terminal CAG/ and the identity of nucleotide -4. Plant Physiol, 1996 Oct, 112(2), 767 - 77 Molecular and biochemical characterization of tomato farnesyl-protein transferase; Schmitt D et al.; The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity . In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified . We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties . Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities . Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro . LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development . Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1 . Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth. Nucleic Acids Res, 1996 Oct 1, 24(19), 3784 - 9 FLP-mediated recombination of FRT sites in the maize genome; Lyznik LA et al.; Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system . The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene . NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment . Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place . The functional FLP gene could be either expressed transiently or after stable integration into the maize genome . The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required . The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA. Biochem J, 1996 Oct 1, 319 ( Pt 1), 109 - 16 Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases; Sanderson SJ et al.; Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly . Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e . 8-12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e . 50-fold) of parent E3 . N-terminal sequence analysis of the truncated 35,000-M(r) protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15,000-M(r) N-terminal fragment comprising both the lipoyl and linker sequences . In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3 . Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2 . Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component. Mol Pharmacol, 1996 Oct, 50(4), 891 - 9 Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding; Jacobitz S et al.; To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations . The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera . Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids . The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily . Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively . {3H}(R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM) . Interestingly, {3H}(R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity . Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM) . In contrast to {3H}(R)-rolipram, {3H}RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM) . Further truncation of the enzyme to Met332-886 had no effect on {3H}RP 73401 binding (Kd = 0.2 +/- 0.03 nM) . We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722 . Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain . As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity. Am J Pathol, 1996 Oct, 149(4), 1221 - 8 Germ cell tumors of the testis overexpress wild-type p53; Guillou L et al.; Several recent studies have suggested that testicular germ cell tumors express high levels of wild-type p53 protein . To clarify and confirm this unexpected result, we have investigated seminomatous and nonseminomatous germ cell tumors at the genomic, mRNA, and protein levels . Thirty-five tumors were examined for p53 overexpression using antibodies directed against the p53 (PAb1801, PAb240, and CM1), mdm2 (IF2), and p21Waf1/Clp1 (EA10) proteins . Thirty-two tumors were screened for p53 mutations by single-strand conformation polymorphism analysis . Eighteen tumors were screened with a functional assay that tests the transcriptional competence of human p53 protein expressed in yeast . On frozen sections, 100, 65, 35, 73, and 0% of tumors reacted with the CM1, PAb240, PAb1801, IF2, and EA10 antibodies, respectively . No p53 mutations were detected by single-strand conformation polymorphism or by functional assay . The fact that many tumors overexpress wild-type p53 but not mdm2 rules out mdm2 overexpression as a general explanation for the presence of wild-type p53 in these tumors . The absence of p21 overexpression suggests that p53 may be unable to activate transcription of critical target genes, which may explain why the presence of wild-type p53 is tolerated in this tumor type, although the mechanism for this transcriptional inactivity remains to be established. J Neurochem, 1996 Oct, 67(4), 1744 - 50 Accelerated myelinogenesis by dietary lipids in rat brain; Salvati S et al.; Our previous work showed an early development of behavioral reflexes in rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n-alkanes (which contain 50% odd-chain fatty acids) in comparison with controls fed a margarine diet . To clarify whether the observed changes might be linked to an early myelination, we have investigated mRNAs involved in myelin synthesis in the brains of offspring at 5 days of age by northern blot and in situ hybridization . Northern blot analysis showed that proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) mRNAs were higher in animals on the lipid diet compared with controls . In situ hybridization with probes specific for PLP, myelin basic protein, and MOG mRNA showed significantly higher numbers of positive cells in test animals compared with controls in all brain regions . This study shows an acceleration of myelinogenesis induced by dietary lipids . These data can give a new insight in the therapeutical approaches involved to promote repair in demyelinating diseases. J Cell Biol, 1996 Oct, 135(1), 85 - 95 Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor; Gould SJ et al.; Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome . We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain . Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold . Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins . We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor. J Cell Biol, 1996 Oct, 135(1), 53 - 61 Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex; Faulstich D et al.; Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles . In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, {COPs} alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits . We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing . delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2 . We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes . A knock-out of the delta-COP gene in yeast is lethal . Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs . Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs . We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11274 - 9 Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought; Jonak C et al.; Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals . We have tested whether MAP kinases are involved in mediating environmental stress responses in plants . Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants . p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein . Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism . Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress . However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa . Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes . Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated . These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10923 - 7 RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways; Grimm S et al.; With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor . Both of these proteins induce apoptosis in mammalian cells . Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis . However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene . By contrast, FADD's ability to induce cell death does not depend on crosslinking . Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD . Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD . Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases. Mamm Genome, 1996 Oct, 7(10), 758 - 66 A radiation hybrid map spanning the entire human X chromosome integrating YACs, genes, and STS markers; Kumlien J et al.; We present a radiation hybrid (RH) map of human Chromosome (Chr) X, using 50 markers on 72 radiation hybrids . The markers, obtained from the consensus map, form a grid spanning the entire chromosome . To check the RH map, the marker order was determined by analysis of presence or absence of retained human DNA fragments in the RHs; the comparison with the consensus showed a similar order . Any STSs, microsatellites, genes, and clones can be positioned and ordered relative to the marker grid . This approach integrates genetic, physical, and large-scale clone mapping and is used to link YAC contigs containing data from various experimental sources. J Infect Dis, 1996 Oct, 174(4), 862 - 6 Immunization with recombinant p17/p24:Ty virus-like particles in human immunodeficiency virus-infected persons; Veenstra J et al.; In studies of the natural history of human immunodeficiency virus type 1 (HIV-1) infection, it has been repeatedly shown that higher-titer antibody responses to the HIV gag p24 protein correlate with less rapid disease progression . In HIV-negative persons, immunization with HIV-1 p17/p24:Ty virus-like particles (p24-VLP) induced humoral and cellular immune responses to p24 . This construct was therefore studied as a potential immunotherapeutic agent with the objective of augmenting the immune response to p24 in a double-blind placebo-controlled trial involving 74 p24 antibody-positive, asymptomatic HIV-1-infected subjects with CD4 cell counts > 350/mm3 . Immunization with p24-VLP was generally well tolerated . Immunization with p24-VLP did not increase p24 antibody levels and had no effect on CD4 cell counts or virus load . The failure to increase p24 antibody titers cannot entirely be explained by the subjects' immunodeficiency because most generated an antibody response to Ty, a yeast component of the immunogen. Arch Biochem Biophys, 1996 Oct 1, 334(1), 104 - 12 Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa; Diczfalusy MA et al.; Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells . However, in yeast little is known about presence and function of acyl-CoA thioesterase activity . Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity . Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE . The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate . YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail . Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested . The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM . The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM) . The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin . Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type . Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with {3H}DFP . p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity . N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases. Cancer Res, 1996 Oct 1, 56(19), 4493 - 8 Early involvement of 6q in surface epithelial ovarian tumors; Tibiletti MG et al.; Complex karyotypes are often seen in primary surface epithelial ovarian tumors (SEOTs) . Conventional cytogenetic as well as fluorescence in situ hybridization analyses coupled with loss of heterozygosity studies identified abnormalities of chromosome 6 as one of the most frequent lesions in these types of tumors . We performed cytogenetic analysis of direct preparations from 40 SEOTs, including borderline tumors and low-, intermediate-, and high-grade carcinomas to verify the frequency of chromosome 6 alterations . We also carried out fluorescence in situ hybridization analysis with a chromosome 6 library and yeast artificial chromosome clones from a region of the same chromosome (6q27) . Chromosome 6 abnormalities were identified in 30 of 32 analyzable SEOTs . Twenty-five of 32 cases showed a deletion of 6q irrespective of their histological grade . We wish to underline that this is the first report proving that del(6q) was the most frequent chromosome anomaly in near-diploid SEOTs and that it was the sole anomaly observed in four SEOTs with diploid complement . Our findings suggest that abnormalities of the telomeric region of chromosome 6 (6q27) may be considered one of the earliest lesions in the pathogenesis of ovarian carcinomas. J Mol Evol, 1996 Oct, 43(4), 384 - 98 Molecular evolution of the 14-3-3 protein family; Wang W et al.; Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes . To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans . The alignment revealed five highly conserved sequence blocks . Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer . Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms . Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis . Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms . Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein . The known invertebrate proteins group with the nonepsilon mammalian isoforms . Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms . Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species . A possible ancestral 14-3-3 sequence is proposed. Hum Genet, 1996 Oct, 98(4), 497 - 9 The gene encoding the p60 subunit of chromatin assembly factor I (CAF1P60) maps to human chromosome 21q22.2, a region associated with some of the major features of Down syndrome; Katsanis N et al.; Trisomy 21 is the most common aneuploidy in humans with a frequency of 1 in 700 live births and is by far the most common defined cause of mental retardation . To analyse which of the chromosome 21 genes is overexpressed early in development-giving rise to the Down syndrome phenotype-and to provide candidate genes for other HSA21 disease loci, we need a transcription map of the chromosome . Therefore, to enrich the gene map of human chromosome 21 we have undertaken a systematic approach to fine mapping and characterising expressed sequences generated by the various cDNA sequencing projects . In this report we show the localisation of the CAF1P60 gene to human chromosome 21 and its fine mapping to 21q22.2 between D21S333 and D21S334 . This mapping position places CAF1P60 in a region of HSA21 which is strongly associated with the major features of Down syndrome . The function of this gene product may have important implications for the phenotype that arises from trisomy 21. Hum Genet, 1996 Oct, 98(4), 460 - 6 YAC and cosmid FISH mapping of an unbalanced chromosomal translocation causing partial trisomy 21 and Down syndrome; Nadal M et al.; Most cases of Down syndrome (DS) result from a supernumerary chromosome 21; however, there are rare cases in which DS is due to partial trisomy of chromosome 21, involving various segments of the chromosome . The characterization of cases of DS that are due to partial trisomy 21 allows the phenotype to be correlated with the genotype . We present a case with features of DS and a partial trisomy of chromosome 21 inherited from a paternal balanced translocation involving chromosomes 13 and 21 . Fluorescence in situ hybridization analysis using yeast artificial chromosome (YAC) probes mapped the breakpoint to 21q22.1, within YAC 230E8, which contains markers CBR, D21S333 and D21S334 . Further mapping using cosmids positioned the breakpoint proximal to CBR . The patient was also monosomic for the distal portion of chromosome 13 (q33-qter) . Many phenotypic features of DS were present including hypotonia, flat occiput, flat facies, up-slanted palpebral fissures, epicanthic folds, flat nasal bridge, macroglossia, open mouth, small ears and a heart murmur . This case further supports the contention that the majority of the phenotypic features of DS map to 21q22-qter and further refines the location of some of them . In addition to the DS phenotype, the patient had a prominent upper maxilla with protruding upper incisors, and low levels of the coagulation factors VII and X, consistent with a syndrome resulting from monosomy 13q33-qter . Since some features overlap between the two syndromes, including severe mental retardation, it is unclear to what extent monosmy for 13q33-qter, trisomy for 21q22.1-qter, or a combination of both, contributed to the common features of the phenotype. Hum Genet, 1996 Oct, 98(4), 454 - 9 A detailed investigation of two cases exhibiting characteristics of the 6p deletion syndrome; Davies AF et al.; Deletions of the short arm of chromosome 6 are relatively rare, only 16 cases having been described in the literature so far . Here we present a detailed investigation by fluorescence in situ hybridisation of two further cases with different but overlapping interstitial deletions involving 6p22, 6p23 and 6p24 . The main features involved are craniofacial malformations, heart and kidney defects, mental retardation/developmental delay, hypotonia and hydrocephalus . By using 36 yeast artificial chromosome and cosmid clones from a contig covering 6p22.3-6p25 and other probes with defined cytogenetic locations within 6p21-6p22 we have precisely localised the breakpoints involved in each of the cases, estimated the sizes of the deleted regions and defined the region that is hemizygously deleted in both cases. Hum Genet, 1996 Oct, 98(4), 443 - 6 Physical linkage and orientation of the human complement C8 alpha and C8 beta genes on chromosome 1p32; Platteborze PL et al.; Human C8 is one of five components (C5b, C6, C7, C8, C9) of the cytolytic C5b-9 complex of complement . It consists of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma), which are encoded in separate genes . Genetic linkage and chromosomal localization studies previously established that C8 alpha and C8 beta are closely linked on chromosome 1p32 . In this study, clones with inserts containing genes for both C8 alpha and C8 beta were isolated from a yeast artificial chromosome (YAC) human genomic DNA library and characterized in an effort to determine intergenic distance and orientation . One clone with a approximately 330-kb insert yielded restriction digest patterns for C8 alpha and C8 beta that agreed with those obtained previously from digests of human genomic DNA, thereby confirming the presence of intact copies of both genes . A second clone with a approximately 280-kb insert yielded similar results; however, it was truncated at the 5' end of the C8 alpha gene . Restriction digests of both clones were subjected to PFGE and Southern blot analysis using probes specific for the terminal exons of C8 alpha (exons 1 and 11) and C8 beta (exons 1 and 12) . Results indicate the genes are physically linked (< 23 kb) and in a 3'-3' orientation . This is the same orientation as the ancestrally related C6 and C7 genes, which are also physically linked on chromosome 5p13. Atherosclerosis, 1996 Sep 27, 126(1), 65 - 75 Inhibition of LDL oxidation and myeloperoxidase dependent tyrosyl radical formation by the selective estrogen receptor modulator raloxifene (LY139481 HCL); Zuckerman SH et al.; Cellular oxidation of protein and lipoproteins is believed to contribute to the pathology associated with both acute and chronic inflammatory processes . Enzymatic, myeloperoxidase and lipoxygenase, and non- enzymatic oxidation of low density lipoprotein, LDL, has been implicated in foam cell formation and the progression of atherosclerotic changes within the arterial wall . In the present study, the in vitro protective role of the selective estrogen receptor modulator, raloxifene, in these oxidant triggered processes has been investigated . Raloxifene, as with estrogen was observed to inhibit both copper mediated LDL oxidation as well as the cellular modification of LDL by murine peritoneal macrophages . Raloxifene was, however, a more potent inhibitor of LDL oxidation than 17 beta-estradiol . The inhibition of macrophage LDL modification by raloxifene was not due to a non-specific effect on all effector functions as phagocytosis of opsonized yeast was comparable with control macrophage cultures . In addition to the protective effects on LDL oxidation, raloxifene also inhibited tyrosyl radical formation catalyzed by myeloperoxidase . The inhibition of myeloperoxidase activity was observed for both the isolated enzyme and in phorbol ester stimulated murine peritoneal neutrophils . In contrast, raloxifene was a weaker inhibitor of horseradish peroxidase . These results demonstrate a potential protective role for raloxifene as an anti-oxidant in in vitro assays designed to evaluate oxidant mediated radical formation and tissue damage. J Biol Chem, 1996 Sep 27, 271(39), 24048 - 54 Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger; Stenmark H et al.; EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol . At its C terminus, the protein contains a cysteine-rich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F . T., Callaghan, J . M., Steele-Mortimer, O., Stenmark, H., Parton, R . G., Campbell, P . L., McCluskey, J., Yeo, J . P., Tock, E . P., and Toh, B . H . (1995) J . Biol . Chem . 270, 13503-13511) . Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif) . Profile-based data base searches identified the FYVE finger in 11 distinct proteins . The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn2+ . Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent . Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPase-deficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization . These results implicate the FYVE finger in the specific localization of EEA1 to endosomes. J Biol Chem, 1996 Sep 27, 271(39), 23653 - 6 Identification of the GalNAc kinase amino acid sequence; Pastuszak I et al.; A new kinase that forms GalNAc-1-P was purified from pig kidney cytosol and identified on gels by labeling with N3-{32P}ATP (Pastuszak, I., Drake, R., and Elbein, A . D . (1996) J . Biol . Chem . 271, in press) . A 50-kDa labeled protein was eluted, digested with trypsin, and the sequences of four peptides representing 49 amino acids showed 90% identity to sequence of human galactokinase reported to be on chromosome 15 . To resolve this dilemma, activities and substrate specificities of galactokinase and GalNAc kinase from human and pig kidney, as well as of galactokinase from the yeast clone transfected with the cDNA from presumptive human galactokinase, were compared . The purified galactokinases phosphorylated galactose, but not GalNAc, whereas GalNAc kinase also phosphorylated galactose when this sugar was present at millimolar concentrations . Extracts of gal 1(-) yeast clone, transfected with presumptive human galactokinase cDNA, had very low galactokinase activity even when yeast were grown on galactose, but good activity with GalNAc . On the other hand, the wild type yeast phosphorylated galactose, but not GalNAc . These data indicate that the sequence reported for galactokinase on chromosome 15 is that of GalNAc kinase, which can phosphorylate galactose when this sugar is present at millimolar concentrations . This transfection thus allows the yeast mutant to grow slowly on galactose-containing media. Mol Gen Genet, 1996 Sep 25, 252(4), 483 - 8 Characterization and application of soybean YACs to molecular cytogenetics; Zhu T et al.; Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes . In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH) . We have screened such a library for low-copy-number clones by hybridization to total genomic DNA . Four clones were chosen for chromosome tagging based upon their low or moderate signal . By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei . FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci . The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes . We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean. J Biol Chem, 1996 Sep 20, 271(38), 23022 - 8 Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase; Fujisawa K et al.; A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N . Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S . (1996) EMBO J . 15, 1885-1893) . p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus . To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using {35S}guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA . Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021 . This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region . These studies localized the minimum Rho binding region to amino acids 934-1015 . To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations . This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding . The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S . (1996) J . Biol . Chem . 271, 13556-13560) and may define a distinct class of Rho effector motif. J Biol Chem, 1996 Sep 20, 271(38), 22941 - 4 FKBP-12 recognition is dispensable for signal generation by type I transforming growth factor-beta receptors; Charng MJ et al.; The FK506-binding protein, FKBP12, is a putative target of type I receptors for transforming growth factor-beta (TbetaR-I) . As the FK506 motif that competes with TbetaR-I for FKBP12 resembles an invariant Leu-Pro dipeptide in TbetaR-I, we replaced Leu193 and Pro194 with Ala, along with mutations across the Gly/Ser box . L193A, P194A, and L193A/P194A do not alter TbetaR-I function; T204D partially activates, independent of ligand; L193A/P194A/T204D was an even more potent constitutive mutation . Association with FKBP12 in a yeast two-hybrid assay was disrupted by P194A, L193A/P194A, and L193A/P194A/T204D, but not L193A or T204D alone . Thus, FKBP12 recognition is dispensable for TGFbeta signaling. EMBO J, 1996 Sep 16, 15(18), 4909 - 18 Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells; Cloutier JF et al.; p50csk is a tyrosine protein kinase (TPK) that represses the activity of Src family TPKs . We previously showed that Csk is a potent negative regulator of antigen receptor signaling in T lymphocytes and that its Src homology (SH) 3 and SH2 domains are required to inhibit these signals . To test the idea that the Csk SH3 and SH2 domains mediate interactions with other cellular proteins, we attempted to identify Csk-associated polypeptides using the yeast two-hybrid system . The results of our experiments demonstrated that Csk physically associates with PEP, a protein tyrosine phosphatase (PTP) expressed in hemopoietic cells . Further analyses revealed that this interaction was mediated by the Csk SH3 domain and by a proline-rich region (PPPLPERTP) in the non-catalytic C-terminal portion of PEP . The association between Csk and PEP was documented in transiently transfected Cos-1 cells and in a variety of cells of hemopoietic lineages, including T cells . Additional analyses demonstrated that the association between Csk and PEP is highly specific . Together, these data indicated that PEP may be an effector and/or a regulator of p50csk in T cells and other hemopoietic cells . Moreover, they allowed