[Brief text messaging (SMS)-delivered smoking cessation intervention for apprentices in vocational schools: a longitudinal intervention study for process and outcome evaluation]. / Kurzintervention zur Förderung der Tabakabstinenz via Short Message Service (SMS) bei Auszubildenden an beruflichen Schulen: Longitudinale Interventionsstudie zur Ergebnis- und Prozessevaluation.

OBJECTIVE: The aim of this study is to test the acceptance and efficacy of an individualised text messaging (SMS)-based intervention to support smoking cessation in apprentices of vocational schools. METHODS: The SMS program MyWay, which generated individualised text messages to support smoking cessation based on an online assessment and a weekly SMS assessment, was developed. The text messages were based on the Transtheoretical Model of Behavior Change and sent to the programme participants over a period of 3 months. The programme was tested in 62 school classes at 6 vocational schools in Bremen, Germany, where 1 086 apprentices participated in an online assessment addressing demographic and smoking related variables. RESULTS: A total of 415 persons (38%) fulfilled the inclusion criteria "daily cigarette smoking", "ownership of a cell phone" and "regular use of text messaging". Of these, 210 persons (51%) participated in the programme. A 6-month follow-up telephone interview for the evaluation of the SMS programme and the assessment of smoking-related variables could be conducted in 125 (60%) of the 210 programme participants. Compared to the baseline assessment, the smoking prevalence (7-day-point prevalence abstinence, 11%) and the intention to stop smoking (p<0.01) were higher at the 6-month follow-up. Furthermore, cigarette craving was lower at 6-month follow-up among persons who still smoked at this point in time (p<0.01). CONCLUSIONS: In comparison to other smoking cessation services, this intervention approach allows us to reach a substantial part of smokers in vocational schools. The text messaging programme is promising for the support of smoking cessation in young adults.

Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention. In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population. Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins. Three classes of missense codons were found: (i) complete loss of function, i.e. mutations; (ii) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.

Intergenic complementation truncation mutants of cyclin-dependent kinase.

The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.

Function of hybrid human-yeast cyclin-dependent kinases in Saccharomyces cerevisiae.

It is now well established that progression through the eukaryotic cell cycle is controlled by oscillations in the activity of cyclin- dependent kinases (CDKs). In many cases, however, the physiological substrate(s) of CDKs are unknown. The Saccharomyces cerevisiae PHO5 gene encodes a secreted acid phosphatase which is induced in response to phosphate starvation. The PHO5 gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation by the products of PHO80 and PHO85. Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer satisfies the biochemical definition of a cyclin/CDK. In the present study, several reporter genes were expressed in S. cerevisiae from promoters which are activated by the transcription factor Pho4p, thereby generating yeast strains which exhibit quantifiable phenotypes that reflect the activity of a specific cyclin/CDK. Positive genetic selections for inhibition of cyclin/CDK function were characterized using the E. coli neo and yeast LEU2 genes. Chromosomal disruptions of the yeast PHO80 and PHO85 genes were constructed and conditions for complementation by plasmid-borne genes were defined. Complementation is achieved at very low levels of expression of both Pho80p and Pho85p. High-level expression of Pho80p results in aberrant PHO5 promoter regulation, characterized by failure to derepress in low-phosphate medium. Genes encoding hybrid CDKs in which regions of Pho85p were replaced with the homologous region of human Cdk2 were constructed, and tested for function in S. cerevisiae by complementation of the pho85 chromosomal gene disruption. Hybrid proteins in which more than two-thirds of the molecule were derived from human Cdk2 retained Pho85p function with respect to high-phosphate repression of the PHO5 promoter. The hybrid proteins require the PHO80 gene product for this function. A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequence which is perfectly conserved in Pho85p and human Cdk2, retains full function. These results demonstrate that, within the context of the conserved structure of CDKs, considerable primary sequence variability can be introduced without loss of the cyclin-dependent function of the CDK.

Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR). As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis. Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism. The system described here may be used for further structure/function analysis of yeast DMR proteins. Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function.

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter.

The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides -676 to -381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides -513 to -501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5' deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 bp sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions -486 to -480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRF1 UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the -676 to -381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.

Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. II. Synthetic Pre-S(2) immunogen.

One purpose of this study was to examine the influence of viral subtype on in vivo antibody production to the pre-S(2) region of the hepatitis B surface Ag. Immunization with hepatitis B surface Ag particles containing the pre-S(2) region of the d or y subtypes identified the B10.M (H-2f) strain as an antibody nonresponder to the pre-S(2) and S regions after immunization with the y subtype, but as an antibody responder to the pre-S(2) and S regions after immunization with the d subtype. Both the S region and pre-S(2) region-specific antibody responses emanated from pre-S(2)/d-specific Th cell function because B10.M mice are T cell nonresponsive to the S region of both subtypes. Although responder/nonresponder status of the B10.M strain was dependent on the pre-S(2) subtype used for immunization, the anti-pre-S(2) antibody produced was totally cross-reactive on both subtypes. This is consistent with the conserved nature of the dominant pre-S(2) antibody-binding site and the highly polymorphic nature of the pre-S(2) sequence that represents the focus of T cell recognition. These data suggest that, to fully benefit from the inclusion of pre-S(2) region sequences, third generation hepatitis B virus vaccines should contain both the d and y subtype sequences of the pre-S(2) region to increase the frequency of pre-S(2) and S-specific antibody responses and to insure Th cell memory relevant to both viral subtypes. A second purpose of this study was to "design" a synthetic pre-S(2) immunogen based on combining the dominant B and T cell recognition sites into a single peptide. A composite peptide consisting of the dominant T cell recognition sequence p151-174 positioned N-terminal to the dominant B cell site p133-143 (i.e., p151-174(133-143] yielded an effective pre-S(2) synthetic immunogen. Interestingly, the orientation of the T and B cell determinants and the context of the T cell site within the larger composite peptide influenced both antibody fine specificity and T cell fine specificity.

Expression of interferon-gamma from hybrid yeast GPD promoters containing upstream regulatory sequences from the GAL1-GAL10 intergenic region.

The expression of human immune interferon (IFN-gamma) is toxic to yeast, resulting in low plasmid stability and copy number. The Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter [Bitter and Egan, Gene 32 (1984) 263-274] has been modified by introduction of upstream regulatory sequences from the yeast GAL1-GAL10 intergenic region [UASG; Guarente et al., Proc. Natl. Acad. Sci. USA 79 (1982) 7410-7414] and utilized to express IFN-gamma. In contrast to the native GPD promoter, the GPD(G) hybrid promoters are regulated by the carbon source. With glucose as the carbon source, a level of expression is observed which is much lower than that obtained with the native GPD promoter. Expression of the hybrid promoters is induced approx. 150- to 200-fold in shaker flask cultures by growth in galactose and similar levels of expression are observed after growth in lactate plus galactose. However, full galactose induction is not observed in the presence of glucose.? Utilization of these regulated promoters has allowed maintenance of plasmids at high copy number with glucose as the carbon source and, after induction with galactose, production of IFN-gamma mRNA at levels more than ten times higher than the native yeast PGK gene transcript. In contrast, the native GPD promoter directs comparable levels of expression when grown in either glucose or galactose resulting in low plasmid copy number and a correspondingly lower IFN-gamma transcript abundance. It is demonstrated that nucleotide sequences more than 240 bp upstream from the TATA box are required for optimal activity of the native GPD promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae.

The cloning and expression of the hepatitis B middle-protein surface antigen gene in the yeast Saccharomyces cerevisiae is described. A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used. Expressed material, in the form of supramolecular particles, was purified and characterized. Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host. This proteolysis was substantially reduced by utilization of a protease-deficient host. Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present. All species were analyzed by N-terminal sequencing after electroelution from gels. Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms. Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types. Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues. The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region.

Hepatitis B vaccine produced in yeast.

A gene encoding the 226 amino acid hepatitis B surface antigen (HBsAg), subtype adw, was cloned into a generalized vector for the expression of heterologous genes in Saccharomyces cerevisiae. The 5' end of the genomic HBsAg gene was replaced with a chemically synthesized DNA segment that conserved the amino acid sequence of the protein but utilized DNA sequences that optimize translation initiation in yeast. High-cell-density fermentations of laboratory strains of Saccharomyces cerevisiae have been developed in which HBsAg production increases linearly with respect to cell mass. The HBsAg is present as a lipoprotein particle in cell lysates and has been purified to homogeneity. The evidence presented indicates that the HBsAg particles may be formed during lysis of the yeast cells. The purified HBsAg particles have a morphology similar to that of the 22 nm particles present in the serum of human chronic carriers of hepatitis B. The reactivity of the yeast-derived HBsAg particles with a series of monoclonal antibodies is essentially identical to that of human plasma HBsAg. By this analysis, therefore, the structure of the HBsAg protein is similar in yeast and in human particles. The purified yeast HBsAg particles were formulated with alum adjuvant and subsequently were shown to confer immunity in chimpanzees to challenge with two heterologous serotypes (adr, ayw) of hepatitis B virus.

Controlled expression and purification of human immune interferon from high-cell-density fermentations of Saccharomyces cerevisiae.

Conditions for high-cell-density fermentations of Saccharomyces cerevisiae strains producing recombinant-DNA-derived proteins were established. Strains producing human immune interferon (IFN-gamma) from the constitutive PGK promoter failed to grow to high cell densities and exhibited low plasmid stability. Regulated expression of IFN-gamma was obtained in similar strains by employing a hybrid yeast GPD promoter that was subject to carbon source regulation due to the presence of regulatory DNA sequences from the yeast GAL 1,10 intergenic region. IFN-gamma expression programmed by this vector was low during growth on glucose and was induced by galactose. Previously defined fermentation conditions employing glucose as a carbon source were applied to this strain, resulting in high ceil densities with higher plasmid stability. Various methods of galactose induction of IFN-gamma expression in high-cell-density fermentations were investigated. Optimal conditions resulted in a 2000-fold induction and production of 2 g IFN-gamma/L fermentation culture.

Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region.

The Saccharomyces cerevisiae secretory process was studied by evaluating secretion efficiency, processing efficiency, and the efficiency of protein folding for hybrid proteins containing the yeast prepro-alpha-factor leader region. Secretion of three proteins, beta-endorphin, calcitonin, and a consensus alpha-interferon (IFN-Con1), were compared in terms of secretion efficiency into the culture medium, beta-Endorphin and calcitonin, both small proteins, were found to be efficiently secreted from logarithmically grown cells. In contrast, the larger IFN-Con1 accumulated in the periplasmic space and cell wall. The glycosylated, unprocessed prepro-alpha-factor/IFN-Con1 fusion protein was also found to be secreted into the culture medium. The presence of (Glu-Ala) dipeptides in the alpha-factor spacer peptide increased the efficiency of cleavage at Lys-Arg in the prepro-alpha-factor/IFN-Con1 protein fusion. Purified secreted IFN-Con1 was structurally characterized to determine the effect of passage through the yeast secretory pathway on the fidelity and efficiency of protein folding. The disulfide structure of the secreted protein was found to be identical with that reported for the native human alpha-interferons.

Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter.

The promoter region from the cloned glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Saccharomyces cerevisiae (Musti et al., 1983) has been characterized. A 653-bp TaqI restriction fragment with a 3' border 24 bp upstream from the ATG initiation codon was isolated and demonstrated to contain all sequences necessary for promoter function in vivo. This DNA segment was converted to a portable promoter by cloning it into M13mp9, and the entire nucleotide sequence of the portable promoter was determined. Two generalized yeast expression vectors have been constructed utilizing the GPD portable promoter. The expression vectors include the yeast 2 mu origin of replication and amplification functions, such that the plasmids are maintained at high copy number in ciro yeast hosts. These vectors direct synthesis of a consensus alpha-interferon (IFN-alpha Con1) as 1% of total cell protein. Hepatitis B surface antigen (HBsAg) was also expressed from these vectors. The 5' end of the HBsAg gene was replaced with a synthetic DNA segment which restored the deleted GPD untranslated leader and utilized optimal yeast codons for the first 30 amino acids. The partially synthetic gene resulted in a 10- to 15-fold increased expression level from GPD vectors yielding HBsAg polypeptide as 2-4% of total cell protein.

Secretion of foreign proteins from Saccharomyces cerevisiae directed by alpha-factor gene fusions.

Fusions between the cloned yeast alpha-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been constructed. The gene fusions encode hybrid proteins that include the first 89 amino acids of the native alpha-factor precursor fused to either a small (beta-endorphin, 31 amino acids) or large (alpha-interferon, 166 amino acids) foreign protein. Proteolytic cleavage sites involved in alpha-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein. The alpha-factor promoter was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins has been characterized by amino acid sequence analysis of the secreted proteins. The proteolytic cleavages involved in the maturation of alpha-factor peptides from the native precursor also occur accurately in the hybrid protein. In addition, cleavages occurred on the carboxyl side of two lysines within the beta-endorphin peptide. Internal cleavages in the interferon protein were also detected. However, in this case, the cleavages occurred at a very low frequency such that greater than 95% of the secreted interferon remained intact.

Purification of DNA-dependent RNA polymerase II from Saccharomyces cerevisiae.

A rapid procedure for the purification of RNA polymerase II from Saccharomyces cerevisiae is described. Total RNA polymerase activity was solubilized from whole cells by sonication in 0.32 M (NH4)2SO4 and RNA polymerase II purified by polyethylenimine fractionation, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, DEAE-Sephadex, and phosphocellulose. The procedure may be completed in 2.5 days and the resultant enzyme is judged to be greater than 90% pure.

Transcription initiation in eukaryotes: analysis of heterologous in vitro systems utilizing components from mammalian and yeast cells.

The properties of heterologous in vitro transcription systems utilizing components from mammalian and yeast cells have been investigated. Purified yeast RNA polymerase II, when supplemented with a full complement of mammalian transcription factors, does not promote specific transcription initiation on cloned mammalian class II genes. Similarly, a complete mammalian transcription system does not initiate specific transcription on cloned yeast class II genes. These results indicate evolutionary divergence in function of yeast and mammalian class II genes and the associated transcription apparatus. The functional differences observed in this study are corroborated by previously reported structural differences between yeast and mammalian RNA polymerase II and class II genes. In contrast, the mechanism of eukaryotic class III gene transcription appears to be evolutionarily conserved. Thus, a mammalian transcription extract specifically transcribes the cloned yeast 5S rRNA gene. This system synthesizes without processing the 130 base 5S rRNA precursor, and this primary transcript may be processed to the mature 120 base RNA using a partially purified yeast processing activity. An homologous yeast class III transcription system has also been developed. This yeast system contains all components necessary for proper synthesis, processing and splicing of yeast tRNA precursors. Using the homologous yeast transcription system, a template in which the 5' flanking region and first 5 base pairs of the 5S rRNA gene had been deleted is utilized to synthesize a 120 base transcript, but the efficiency of transcription is reduced to about 10% that of the wild type gene. Thus, the yeast 5S rRNA gene has an internal transcription control region, but the immediate 5' flanking sequence have effects on the level of transcription.

Transcription of viral genes in chromatin from adenovirus 2 transformed cells by exogenous eukaryotic RNA polymerases.

The transcription of chromatin from adenovrius 2 transformed rat cells by murine plasmacytoma RNA polymerases I, II and III has been studied. Both the total RNA synthesis and transcription of the integrated adenovirus 2 genes by RNA polymerase II represent de novo DNA transcription as assessed by their sensitivity to actinomycin D. It is shown that each RNA polymerase class has characteristic ionic strength activation profiles and metal ion requirements. RNA polymerase II transcribes the integrated adenovirus 2 genes in chromatin at a frequency 25- to 50-fold higher than their sequences are represented in the genome. In contrast, no detectable viral RNA is synthesized when deproteinized DNA is transcribed. In the presence of Mn2+, all three RNA polymerases (I, II and III) transcribe the integrated viral genes at approximately the same relative frequencey. However, the Mg2+ as divalent cation, the proportion of the total RNA which represents viral gene transcripts is increased 3- to 4-fold with RNA polymerase II, while it remains unchanged for RNA polymerases I or III.