The detection of simulated malingering using a computerized priming test.

A word completion priming test was used to differentiate between normal student control subjects and students instructed to malinger. Controls (n = 60) were instructed to do their best, while malingering subjects (n = 60) were instructed to fake a memory deficit for credit and possible financial compensation. Subjects initially rated and completed stems for words that had at least 10 possible completions. Thirty minutes later, subjects rated and completed stems for words that were either uniquely defined by the stem or could only be completed with a variation of the word. Simulated malingerers and controls differed significantly on response latencies (time to produce rated words-time to produce baseline words, 10 second time limit) and priming scores. Discriminant function analyses showed that as high as 92% of the controls could be correctly identified, and 73% of the malingerers could be correctly identified. These results indicate that priming tests can be used in the detection of malingering.

Properties and engineering of a mutant STA promoter of Saccharomyces diastaticus.

A new allelic variant of the STA2 gene of S. diastaticus, designated as STA2K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region of STA2K is described, with an emphasis on its peculiar structural feature: A 1.1-kb natural deletion located 189 nucleotides upstream of the translation start codon. The strength of the STA2K promoter was found comparable to that of known strong constitutive yeast promoters (ADH1, GAPDH). Regulated glucoamylase expression was demonstrated by chimeric promoters, which were constructed by placing the STA2K promoter under the control of either the PHO5 or CYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASPHO5-STA2K chimeric promoter by phosphate depletion resulted in a destructive overexpression of the secreted glucoamylase, which completely halted cell growth, and promoted cell decay. In contrast, UASCYC1 was shown to mediate a fine-tuned regulation both by glucose concentration and, indirectly, by starch, the substrate for the glucoamylase to produce glucose.

Cloning of a new allelic variant of a Saccharomyces diastaticus glucoamylase gene and its introduction into industrial yeasts.

A new allelic variant of the STA2 gene, designated as STA2K, coding for a secreted glucoamylase, was cloned. Differences were revealed both in the structural gene and in the promoter region, as compared to other STA genes. The most peculiar structural features of STA2K are 1. a 1.1-kb natural deletion in its promoter located 189 nucleotides upstream of the translation start codon; and 2. an Asn-->Asp single amino acid change within the putative active site of the encoded glucoamylase. Neither the presence of glucose in the medium nor the host cell's mating type constellation affected the expression level of STA2K in S. cerevisiae. Self-replicating yeast plasmids containing STA2K were constructed and used to transform a laboratory yeast strain and various brewing strains. Pilot brewing tests with glucoamylase-secreting transformants of a brewing strain produced superattenuated beers at accelerated fermentation rates.

The sequence and potential regulatory elements of the HEM2 promoter of Saccharomyces cerevisiae.

This paper reports the 1890-bp sequence located upstream of the HEM2 gene of Saccharomyces cerevisiae. The following potential regulatory protein-binding motifs were found: ABF1-binding site, yAP1-binding site, two REB1-binding sites, a cyclic AMP-responsive element, RAP1-binding site, and several HAP2-HAP3-HAP4 binding sites, implicating a complex regulatory mechanism governing expression for the HEM2 gene.

Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae.

A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.

Construction of versatile Escherichia coli--yeast shuttle vectors for promoter testing in Saccharomyces cerevisiae.

The promoterless PHO5 gene of the yeast Saccharomyces cerevisiae, encoding the repressible acid phosphatase (AP) was utilized as a reporter gene for the construction of a novel vector system for selection and functional analysis of yeast promoters. The Escherichia coli-yeast shuttle plasmids, pZHB81 and pZHB82, contain different arrays of unique restriction sites located upstream of the PHO5 coding region. Yeast promoters could be screened from random DNA fragments (cloned in the upstream sites) for their ability to direct the expression of the PHO5 gene in transformed (AP-deficient) yeast host cells. AP-expressing transformants were selected directly on agar plates by using a routine colony staining method. Relative promoter strength was assessed by direct assay for AP activity in cell lysates.

Isolation and characterization of nuclear hnRNP complexes from Drosophila melanogaster tissue culture cells.

Fractionation on sucrose gradients of nuclear described extracts prepared from cultured Drosophila melanogaster cells by sonication of the nuclei in the presence of rat liver cytosol RNAase inhibitor revealed a complex polysome-like pattern of nuclear ribonucleoprotein complexes. The bulk of these heterogeneous ribonucleoprotein (hnRNP) complexes sedimented in the 30S to 80S zone of the sucrose gradient. According to biochemical and morphological data, the monomer particle proved to be the 45S hnRNP and its average diameter was found by electron microscopy to be 24-26 nm. The buoyant density of both the mono and polyparticles was about 1.4 g/cm3, with a slight degree of heterogeneity. The proteins from different zones of the sucrose gradient were composed primarily of similar polypeptides of 47 000, 56 000, 64 000, 96 000 and 130 000 daltons. Complete dissociation of nuclear hnRNP complexes was observed by resedimentation of the particles in the presence of 0.7 M NaCl or 4 M urea. RNAase A digestion (0.1 microgram/ml at 0 degree C for 10 min) resulted in the solubilization of part of the hnRNP and aggregation of some particles. The bulk of the RNA isolated from the different sized hnRNP complexes sedimented in the 7 to 11S region in the sucrose gradient. The large hnRNP complexes contained hnRNA strands larger than 15S, up to 28S. The base composition of the RNA from the 45S monoparticles proved to be AU type: A + U/G + C = 1.7. The RNAs from the 60-75S and 90- 100S polyparticles were also AU type, with an A + U/G + C ratio of 1.46 and 1.21, respectively. The hnRNP complexes exhibited marked heterogeneity in the electron microscope. Our biochemical and morphological observation point to a nonrandom organization of hnRNP particles in Drosophila melanogaster nuclei.

Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants.

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.

Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA.

A selective plaque assay that uses thymidine kinase (TK)-deficient human 143 cells was developed to titer mixtures of TK(+) and TK(-) vaccinia virus. With this assay it could be shown that methotrexate-resistant TK(+) virus was formed in cells coinfected with TK(-) virus and wild-type virus DNA. By substituting vaccinia DNA fragments cloned in plasmids for virion DNA, this marker rescue system provided the basis for mapping the TK gene. Of the 15 HindIII fragments, only J could rescue five independently derived TK(-) mutants. This 5000-base-pair (bp) fragment maps approximately 80,000 bp from the left-end of the 180,000-bp vaccinia genome. Marker rescue could be detected with 18 ng or less of plasmid and was proportionate to DNA concentration. The resistance to methotrexate of the TK(+) recombinants was shown to be due to TK synthesis. Evidence that the HindIII J fragment contains the structural TK gene and not a regulatory element was demonstrated by the synthesis of active TK in a cell-free system programmed with mRNA selected by hybridization to the plasmid. Previous studies [Belle-Isle, H., Venkatesan, S. & Moss, B. (1981) Virology 112, 306-317] indicated that mRNAs coding for three immediate early polypeptides with molecular weights of 41,000, 21,000, and 17,000 map within HindIII J. The mapping of the easily selectable vaccinia virus TK gene now opens the way to genetic manipulations that should increase our understanding of vaccinia virus gene expression and facilitate the use of vaccinia virus as an efficient cloning vector for foreign genes.

HnRNP particles from Drosophila melanogaster cells.

The isolation and characterization of HnRNP from cultured Drosophila melanogaster cells is described. HnRNP particles were extracted from the purified nuclei of sonication in the presence of rat liver cytosol RNAse inhibitor. The nuclear extract was centrifuged on a 15-30% sucrose gradient. The main part of the heterogeneous HnRNP material was localized in the 30 to 80S region of the sucrose gradient. According to the results of re-sedimentation studies the monomer particle was 45S. The buoyant density of HnRNP particles from different regions of the sucrose gradient were equal to approximately 1.4. The protein composition of the particles was analyzed by urea-SDS-polyacrylamide gel electrophoresis. There are five main and a few minor bands. Only the main polypeptides have a slightly higher molecular weight than those of the major polypeptides of 30S subparticles from rat liver nuclei. According to electron = microscopic studies the particles are heterogeneous and the average diameter was found to be 24-26 nm both on the basis of negative contrast and platinum-palladium shadowed pictures.

Pattern of segregation of chicken HPRT phenotype in Chinese hamster-chick red blood cell hybrids.

The pattern of segregation of hypoxanthine phosphoribosyltransferase (HPRT, E.C. 2.4.2.8) was determined in synchronized Chinese hamster-chick red blood cell hybrids. Three hybrid lines were synchronized at the G1-S boundary. Bromodeoxyuridine pulses were subsequently applied throughout the S phase, and the frequency of the segregant clones was determined. It was found that the segregation of the chicken-specific HPRT phenotype associated with the loss of a chromosome was potentiated by bromodeoxyuridine administered during the first hour following release of the block.

Cleavage of pre-mRNA sequences by ribonucleases bound to nuclear RNP particles of rat liver.

The 30S nuclear RNP particles from rat liver have been shown to split the double-stranded- (ds) and single-stranded (ss) sequences of nuclear pre-mRNA. Experiments performed in vitro have demonstrated that 1) a 5'-exonuclease and an endonuclease specific for double-stranded pre-mRNA sequences exist in the 30S pre-mRNP particles; 2) in dsRNA monophosphorylated 5'-termini arose in the course of incubation with 30S RNP and most of the products remained double-stranded. The analysis of terminal pNp nucleotides revealed a relatively high ratio of pPyp in the cleaved dsRNA, whereas the nucleosides in 5'-terminal pNp of ssRNA showed nearly random distribution. Our results provide a possible explanation for the appearance of pNp termini during the processing of nuclear pre-mRNA of mammalian cells.

Methylated cap formation by enzymes bound to nuclear informofer particles.

Rat liver nuclear 30 S ribonucleoprotein particles containing pre-mRNA and nuclear sap proteins have been shown to modify in vitro the synthetic dinucleotide ppGpC in the presence of GTP and S-adenosyl-L-methionine (SAM) by the formation of a blocked and methylated (capped) structure 7(meG(5')ppp(5'-GmepC. In the absence of SAM the predominant reaction was GpppGpC. Our results indicate that the 30S ribonucleoprotein particles (informofers) as well as the proteins of the nuclear sap possess both guanylyltransferase, N7-, and 2-o-methyltransferase activities.

The effect of bromodeoxyuridine on the segregation of the chicken-specific HPRT gene from Chinese hamster-chick red blood cell somatic hybrids.

The segregation of the chick-specific HPRT gene was studied in three Chinese hamster-chick red blood cell hybrid lines. The three lines showed individual segregation kinetics, the segregation taking place in an exponential-like fashion. Bromodeoxyuridine becomes incorporated into the nuclear DNA and increases the spontaneous segregation rate.

On the nature of 5' termini in nuclear pre-mRNA of Ehrlich carcinoma cells.

5' terminal nucleosides of nuclear pre-mRNA of Ehrlich ascites carcinoma cells were analyzed by a combination of different chromatographic methods and phosphatase treatment. The heavy nuclear pre-mRNA contains mainly unblocked triphosphorylated nucleosides at the 5' end, although some capped 5' ends could also be found. In this respect, it differs from cytoplasmic poly(A)+ mRNA which contains blocked 5' termini and no triphosphorylated ends. The 5' terminal nucleotides in pre-mRNA are pppGp and pppAp (in a ratio of 3:2). The determination of pppNp content in poly (A)+, poly(U)+, and poly (A)-(U)- fragments of RNA has been used as an approach to establish the topography of pre-mRNA. We also established that the technique for isolation of triphosphorylated 5' terminal fragments of RNA based on hydroxyapatite chromatography (Bajszár, Samarina, and Georgiev, 1974) is still valid in the presence of blocked oligonucleotides. The latter do not interfere with fragments containing free triphosphate groups. Using this technique, we showed that a small but significant portion of triphosphorylated 5' end fragments of 100 nucleotides in length contain oligo(U) sequences reacting with poly(A)-Sepharose.

The properties of oligonucleotide fragments containing the triphosphorylated 5'-termini of nuclear pre-mRNA from Ehrlich ascites carcinoma cells.

Triphosphorylated 5'-end fragments 50-150 nucleotides in length were isolated from nuclear pre-mRNA with the aid of a hydroxyapatite chromatography. They are enriched in U and G (28 and 26%, respectively). About 15% of the fragments isolated from poly(U)+RNA contain poly(U) tracks. Neither poly(A)- nor double-stranded sequences were found. Hybridization experiments in conditions of vast DNA excess demonstrated that the 5'-end fragments contain a low amount of highly repetitive sequences but enriched in sequences hybridizing at C0t 1/2 approximately 100.

Effect of human adenoviruses on the response of chickens to sheep erythrocytes.

Human adenovirus types 6, 8, and 12 were immunosuppressive in chickens. A single intravenous injection of adenoviruses markedly depressed the 19S hemolytic plaque-forming cell response in the spleen to the immunization with sheep red blood cells. Hemagglutinin production was also decreased in adenovirus type 6-infected chickens. Adenoviruses caused a transient immunosuppression in chickens which could be detected 2 to 3 days after the virus infection, and no depressive effect was found 16 to 20 days after virus injection. The possible mechanism of immunosuppression observed is discussed.

Inverted repetitions in mammalian DNA transcribed into nucleus-restricted hairpin-like structures of pre-mRNA.

Hairpin-like structures isolated from giant pre-mRNA are the transcripts from inverted repetitions of DNA.