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Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB-231, MDA-MB-436, BT549, MCF-7ADR, Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting areA gene.

Genetic studies have indicated that the facB gene of Aspergillus nidulans is a major regulatory gene involved in acetamide and acetate utilisation. Sequencing of the facB gene revealed that it encodes a protein that contains an N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster for DNA binding, leucine zipper-like heptad repeat motifs and central and C-terminal acidic alpha-helical regions, consistent with a function as a DNA-binding transcriptional activator. The Zn(II)2Cys6 cluster shows strong similarity with those of the Saccharomyces cerevisiae carbon metabolism regulatory proteins CAT8 and SIP4. A significant level of similarity with CAT8 is found throughout the length of the protein, suggesting at least partial functional homology. The facB genes of Aspergillus oryzae and Aspergillus niger were also sequenced and found to be highly conserved. Deletion of the facB gene confirmed that it is required for growth on acetate as a sole carbon source. Functional dissection using deletion and fusion constructs and in vitro mutagenesis indicated that the Zn(II)2Cys6 cluster and the C-terminal end of the protein are required for function.

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level of amdS expression. Mobility shift studies have identified a factor in A. nidulans nuclear extracts which binds to this CCAAT sequence. In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed an A. nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designated hapC, with extensive homology to HAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, it has been shown that the hapC deletion results in reduced levels of expression of an amdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT binding activity to the amdS or gatA promoters, indicating that hapC may encode a component of the complex binding at this sequence.

There has been renewed interest in the idea that attentional dysfunction may underlie autistic symptomatology (e.g., Bryson, Wainwright-Sharp, & Smith, 1990; Dawson & Lewy, 1989a, 1989b). Existing research indicates problems with overfocused attention (Lovaas et al., 1971; Rincover & Ducharme, 1987), and with shifting attention between sensory modalities (Courchesne et al., 1990). These phenomena were examined further by using Posner's (1978) visual orienting task with a group of high-functioning autistic adolescents and adults, and matched normal controls. Our results indicate that autistic people have difficulty processing briefly presented cue information. Evidence of problems disengaging and shifting attention within the visual modality was also provided. The findings can be seen as consistent with previous behavioral, autonomic, and electrophysiological research which has revealed impairments in the registration, processing, and response to external stimuli.

Pigeons were given a choice between two identical-duration situations (terminal links of chain schedules). One terminal link of the choice pair provided two food deliveries, and the other provided five. The exact times of these food deliveries differed between the terminal links and were varied over conditions. A single response during the initial link gave immediate access to the corresponding terminal link. Forced trials, during which only one of the initial-link keys was lighted, were interspersed with choice trials during which both initial-link keys were lighted. Choice tended to favor whichever terminal link was correlated with the higher sum of the immediacies (i.e., the sum of the reciprocals of the delays to each of the reinforcers following the choice, with all delays measured from the choice). Latencies on forced trials and on choice trials also were related (negatively) to the sum of the immediacies. This correlation among response measures (choice and latencies) suggests that both measures are manifestations of the effect of conditioned reinforcement on response tendencies.

Kinetic analyses of runoff transcription in a cell-free eucaryotic transcription system revealed that the bacteriophage lambda 4S RNA terminator caused human RNA polymerase II to pause on the template and partially terminate transcription of transcripts initiated by the adenovirus 2 major late promoter. Analogous to the procaryotic RNA polymerase, the eucaryotic enzyme terminated just beyond the guanine-plus-cytosine-rich region of dyad symmetry in the terminator sequence. These results suggest that the eucaryotic RNA polymerase II may respond to transcription termination sequences similar to those used by the procaryotic enzyme. However, similar templates containing lambda tint or lambda tR1 terminators did not elicit pausing or termination, suggesting that other features, such as sequence specificity, may also be involved.

Subdural collections in infants are common complications of meningitis, trauma and post surgical procedures. Careful ultrasonic examination via the anterior fontanelle is a reliable method of demonstrating these collections. It has an accuracy comparable to computed tomography and is our first line of investigation.

We have investigated the specific DNA and RNA requirements for rho-dependent transcription termination in vitro. As a model, we have used templates containing the rho-dependent terminator of the Escherichia coli trp operon, trp t'. Templates containing the trp t' region direct specific rho-dependent termination in vitro, with concomitant stimulation of the rho NTPase activity, and deletion of the trp t' region results in templates that do not induce rho-dependent termination or rho NTPase activity. Addition of ribonuclease T1 to transcription reactions specifically eliminated transcription termination and rho NTPase activity. These results demonstrate the requirement for a specific RNA component within the trp t' transcript necessary for NTPase activation and rho-dependent transcription termination. Active transcription is not a prerequisite for rho NTPase activation; trp t' RNA (rho-terminated transcripts) and read-through transcripts, which contain the trp t' region, activated the rho NTPase when rho was added after inhibition of transcription. As is true for synthetic polynucleotides known to activate the rho NTPase, the trp t' region has few G residues. This reduces the potential for the formation of stable secondary structures in the RNA transcript, and may be one determinant of sites specifying rho-dependent termination of transcription. The implications of this are discussed in the light of the lack of significant sequence homologies between rho-dependent transcription termination sites.

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5' ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.

A study of the initial velocity kinetics of the Escherichia coli transcription termination protein rho, with respect to its poly(C)-dependent ATPase, indicates that this reaction occurs by an ordered sequential mechanism. Product inhibition and substrate analogue studies suggest that ATP binding must precede the binding of poly(C) and that the order of release of the products is ADP followed by Pi, then poly(C). A possible mechanism for relating the ATPase to the termination reaction of rho is discussed in relation to the model for rho proposed by Richardson (5).

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.

We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.

We have determined the complete nucleotide sequence of the thymidine kinase (ATP:thymidine 5' phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus type 1 strain CL101 from a plasmid clone of viral DNA derived by Enquist et al. [Enquist, L. W., Vande Woude, G. F., Wagner, M., Smiley, J. R. & Summers, W. C. (1979) Gene 7, 335-342]. A cDNA copy of the 5' end of thymidine kinase mRNA was also analyzed to locate the transcribed sequences. The transcribed portion of the gene is approximately 1300 nucleotides in length and appears to contain no intervening sequences. There is an untranslated region of 107 nucleotides at the 5' end of the mRNA followed by an open reading frame of 1128 nucleotides. The gene is thus capable of coding for a protein of 376 amino acids. Sequences similar to those thought to be involved in control of transcription and translation of a variety of eukaryotic and viral genes such as a "Hogness box" and A-A-T-A-A-A polyadenylylation signals are also present in the herpesvirus thymidine kinase gene.

The presence of a chloroform-methanol extract of cat brain in a carbon tetrachloride phase separating two aqueous phases resulted in an increased passage of [3H]choline across the organic phase which was inhibited by the choline transport inhibitor hemicholinium-3 and by high concentrations of non-radioactive choline. In the absence of cat brain extract, [3H]choline passage across carbon tetrachloride was neither inhibited by hemicholinium-3, nor by non-radioactive choline.

The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide 'mapping' and N-terminal analysis.

Synchronized single-round transcriptions, in vitro, from templates encoding the leader RNA of the ilvB and ilvGEDA operons result in the accumulation of a transcript consistent with RNA polymerase pausing after the 1:2 stem that could form in each of the leader RNAs. Addition of L-factor or guanosine 5'-diphosphate,3-diphosphate extended the pause half-life obtained with the ilvB template; addition of L-factor and guanosine 5'-diphosphate,3'-diphosphate together had an additive effect on the pause half-life. L-factor also extended the pause half-life of the pause obtained with the ilvGEDA template; however, addition of guanosine 5'-diphosphate,3'-diphosphate did not. The results obtained are consistent with the model for attenuation proposed by Yanofsky et al. (Yanofsky, C., Das, A., Fisher, R., Kolter, R., and Berlin, V. (1984) UCLA Symposium of Gene Expression (Hamer, D., and Rosenberg, M., eds) pp. 295-310, Alan R. Liss, Inc., New York) in which transcriptional pausing after the synthesis of the 1:2 stem-loop closely couples transcription and translation of the leader region.

The effects of Al3+, Cd2+ and Mn2+ on human erythrocyte choline transport, Na-K-ATPase, Ca-Mg-ATPase and intracellular K+ levels were examined. The concentrations used were below the levels which caused significant haemolysis (less than or equal to 300 microM). All three cations inhibited concentrative choline accumulation over 3 hr [IC50 values at 1 microM choline were 35 microM (AlCl3), 250 microM (CdCl2) and 300 microM (MnCl2)] but at the concentrations tested, none decreased initial rates of choline uptake. The effects of Cd2+ and Mn2+ (but not Al3+) on choline accumulation were reversed by removing the cations from the extracellular medium by washing. All three cations also inhibited efflux of choline, at 1 microM choline, 30% inhibition being produced by 33 microM AlCl3, 81 microM CdCl2 and 111 microM MnCl2. At subhaemolytic concentrations, only CdCl2 inhibited Na-K-ATPase, (IC50 = 147 microM) and none of the cations significantly inhibited Ca-Mg-ATPase. Intracellular K+ levels were only reduced by the highest concentration of AlCl3 used (100 microM). These results suggest that inhibition of choline accumulation and efflux in erythrocytes by Al3+, Cd2+ and Mn2+ is not explicable solely in terms of either inhibition of Ca-Mg-ATPase, or inhibition of Na-K-ATPase causing reduced intracellular K+. Our conclusions are similar to those previously obtained using synaptosomes and provide support for the hypothesis that inhibition of choline transport by Al3+ may contribute to a number of disease states.

We identified in herpes simplex virus type 1-infected cells six cytoplasmic transcripts which were complementary to BamHI restriction endonuclease fragment Q. Two transcripts appeared in major amounts compared with the other four. One major transcript of about 1.4 kilobases was the mRNA for the viral thymidine kinase, was synthesized at intermediate times, and was classified as a beta transcript. The other major transcript was synthesized at late times and was classified as a gamma transcript. This late transcript was about 3 kilonucleotides long and was transcribed in the same direction as the gene for thymidine kinase. The 5' end of this late RNA was located by RNA sequence analysis and was 23 nucleotides downstream from the polyadenylation site for the thymidine kinase mRNA. This finding led to the conclusion that the control region for the 3-kilobase gamma transcript is contained within the 3' untranslated region of the thymidine kinase transcript.

On the basis of observations of adenosine-Ca2+ competition, we assessed the effects on erythrocyte adenosine transport of Ca2+ channel antagonists, mono- and divalent cations, and Cl- and Cl- transport inhibitors. The Ca2+ channel antagonists, diltiazem and verapamil, competitively inhibited adenosine influx (Ki = 158 +/- 17.4 and 13.5 +/- 1.3 microM at 10 microM adenosine, respectively), despite no apparent effect on transport by Ca2+, Mg2+, Na+, or K+. Verapamil also inhibited uridine efflux (Ki = 1.7 +/- 0.3 microM at 84-100 microM intracellular uridine). The absence of Cl- decreased adenosine influx rates from 0.615 +/- 0.013 to 0.386 +/- 0.008 nmol X s-1 X ml intracellular H2O-1. The Cl- transport inhibitors, diisothiocyanostilbene disulfonate (10 microM), furosemide (1 mM), and NO-3 (145 mM), decreased adenosine influx rates to 0.301 +/- 0.008, 0.325 +/- 0.013, and 0.430 +/- 0.009 nmol X s-1 X ml intracellular H2O-1, respectively. These studies indicate that the Ca2+ channel antagonists inhibit adenosine release and uptake and therefore may modulate adenosine-mediated events. Additionally, they suggest that adenosine and anion transport systems are linked or share common features.