Production of human parvovirus 4 VP2 virus-like particles in yeast and their evaluation as an antigen for detection of virus-specific antibodies in human serum.

BACKGROUND: Human parvovirus 4 (PARV4) is a recently discovered member of the Parvoviridae family, which is not closely related to any previously discovered human parvoviruses. PARV4 has been isolated from the plasma of individuals with symptoms of acute viral infection; however, until recently PARV4 had not been associated with any disease, and its prevalence in the human population is yet to be established. METHODS: The major capsid protein VP2 of PARV4 was generated in the yeast Saccharomyces cerevisiae and used for serological detection of virus-specific IgG and IgM in the sera of low-risk individuals. RESULTS: One hundred and seventy serum specimens obtained from patients with acute respiratory diseases were tested for PARV4-specific IgG and IgM antibodies. Sixteen individuals (9.4%) were diagnosed as seropositive, including 6 IgM and IgG positive, 6 IgM positive/IgG negative and 4 IgG positive/IgM negative. Seven of the 16 seropositive individuals were between the ages of 3 and 11 with no evidence of parenteral exposure to PARV4 infection. CONCLUSION: Our data demonstrate that recombinant yeast-derived VP2 protein, self-assembled to virus-like particles, can represent a useful tool when studying the seroprevalence of PARV4 infection. The presence of PARV4-specific antibodies in a low-risk group may indicate the possibility of alternative routes of virus transmission.

Hantavirus infection among wild small mammals in Vellore, south India.

Wild indigenous small mammals including 83 rodents (bandicoot and black rats, and house mice) and a shrew captured from multiple sites in Vellore, south India, were tested for serological and molecular evidence of hantavirus infection. Indirect immunofluorescence assay (IFA) using Hantaan virus (HTNV) antigen indicated hantavirus-reactive antibodies in 16 (19.3%) of 83 rodents (bandicoot and black rats). Western blot (WB) using Thailand virus (THAIV) antigen confirmed hantavirus-reactive antibodies in nine of the 16 HTNV IFA-positive rodents. Reverse transcription polymerase chain reaction (RT-PCR) of lung and kidney tissue of captured mammals resulted in the detection of partial S segment sequence in a bandicoot rat. This study complements our earlier reports on hantavirus epidemiology in south India and documents first laboratory evidence for rodent-associated hantaviruses in south India.

Seroprevalence of SV40-like polyomavirus infections in captive and free-ranging macaque species.

BACKGROUND AND METHODS: To investigate the seroprevalence of polyomavirus infections in macaques, we analyzed 1579 sera from nine different species for antibodies cross-reactive with simian virus 40 (SV40) in an enzyme-linked immunosorbent assay. Most samples were collected from captive animals, but we also investigated a colony of free-ranging Barbary macaques (Macaca sylvanus). RESULTS: High seropositive rates were found in rhesus macaques (Macaca mulatta; 74.7%), cynomolgus macaques (Macaca fascicularis; 44.8%) and Tonkean macaques (Macaca tonkeana; 41.7%), especially in animals imported from China. Low rates were measured in cynomolgus macaques from Mauritius (8.8%), and in Barbary macaques (1.4%). Seropositivity was age-dependent increasing to >70% in animals of 5 years and older. CONCLUSIONS: High seroprevalence rates were found in different species of macaques, dependent on their origin. Very low infection rates found in Barbary macaques and cynomolgus macaques from Mauritius suggest that these animals in the wild are not commonly infected by SV40-like viruses.

Mapping of B cell epitopes in measles virus nucleocapsid protein.

The B-cell response against measles nucleoprotein (MeN) plays an important role in the control of measles infection. However, the data on B cell epitopes of MeN are still limited. The objective of this study was to identify B cell epitopes in MeN using monoclonal and polyclonal antibodies raised against recombinant yeast-expressed MeN (rMeN) as well as human sera from measles-positive individuals. After immunization of mice, 15 monoclonal antibodies (mAbs) against rMeN were generated. The B cell epitopes were localized using recombinant overlapping MeN fragments, PepScan analysis, and competitive ELISA. The epitopes of 14 mAbs were mapped within the C-terminus of MeN between amino acids (aa) 419 and 525. Four mAbs recognized a linear epitope located within a sequence of aa 440-448. Competitive ELISA revealed a cluster of conformational mAb epitopes. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals. Thus, the majority of B cell epitopes are located at the C-terminal domain of MeN. These findings provide new data on the antigenic structure of MeN and are in agreement with recent experimental evidence indicating that the C-terminal domain of MeN is well accessible on the surface of nucleocapsid-like structures.

Size and position of truncations in the carboxy-terminal region of major capsid protein VP1 of hamster polyomavirus expressed in yeast determine its assembly capacity.

The hamster polyomavirus major capsid protein VP1 was modified in its carboxy-terminal region by consecutive truncations and single amino acid exchanges. The ability of yeast-expressed VP1 variants to form virus-like particles (VLPs) strongly depended on the size and position of the truncation. VP1 variants lacking 21, 69, and 79 amino acid (aa) residues in their carboxy-terminal region efficiently formed VLPs similar to those formed by the unmodified VP1 (diameter 40-45 nm). In contrast, VP1 derivatives with carboxy-terminal truncations of 35 to 56 aa residues failed to form VLPs. VP1 mutants with a single A336G aa exchange or internal deletions of aa 335 to aa 346 and aa 335 to aa 363 resulted in the formation of VLPs of a smaller size (diameter 20 nm). These data indicate that certain parts of the carboxy-terminal region of VP1 are not essential for pentamer-pentamer interactions in the capsid, at least in the yeast expression system used.

Generation of monoclonal antibodies of desired specificity using chimeric polyomavirus-derived virus-like particles.

Foreign protein sequences presented on hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic. The current study was aimed to evaluate VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. Chimeric VLPs carrying a 9 amino acid (aa)-long cytotoxic T-cell epitope (STAPPVHNV) of human mucin 1 (MUC1) elicited a strong epitope-specific humoral immune response in mice and promoted the production of MUC1-specific mAbs. From a total of seven mAbs of IgG isotype generated against the chimeric VLPs, two mAbs were directed against the MUC1 epitope and five mAbs against the VP1-carrier. Two out of five anti-VP1 mAbs recognized epitopes located at the previously defined insertion site #2 (aa 223/224), which confirms its surface-exposed localization. Chimeric VLPs carrying a 120-aa long sequence of Puumala hantavirus (PUUV) nucleocapsid protein (NP) promoted the generation of five mAbs of IgG isotype specific to PUUV NP. All mAbs recognized the full-length NP of different PUUV strains. In contrast, no VP1-specific mAbs were obtained. The ability of chimeric VLPs to activate antigen-presenting cells was evaluated by studying the uptake of chimeric VLPs by murine spleen cell-derived dendritic cells (DCs). Efficient uptake of VLPs and activation of murine DCs were demonstrated, which may represent the basis of the strong immunogenicity of chimeric VLPs. In conclusion, chimeric VLPs effectively stimulated the production of IgG antibodies specific for foreign epitopes presented at surface-exposed regions. Thus, chimeric HaPyV VP1-derived VLPs represent efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins.

Detection of human hantavirus infections in Lithuania.

BACKGROUND: In Europe certain hantaviruses are known to cause hemorrhagic fever with renal syndrome of different severity. The objective of the present investigation was to study the presence of hantavirus infections in Lithuania. MATERIAL AND METHODS: Two different serum panels from cancer patients (n = 438) and blood donors (n = 299) from Lithuania were tested by monoclonal antibody capture IgG ELISA using yeast-expressed recombinant nucleocapsid (rN) proteins of Puumala virus (PUUV), Hantaan virus (HTNV) and Dobrava virus (DOBV). The reactivity of ELISA-positive sera was proven in Western blot tests using various hantavirus rN proteins. Selected serum samples were further analyzed by focus reduction neutralization assays. RESULTS: In the IgG ELISA 39 sera from the cancer patients and four sera from blood donors were found to be reactive with at least one of the rN proteins. By immunoblot using the three yeast-expressed rN proteins, the ELISA reactivity of 36 of 39 and two of four serum samples from cancer patients and blood donors, respectively, was confirmed; this corresponds to a seroprevalence of 8.2% and 0.7%, respectively. In ELISA, the majority of the samples reacted exclusively with rN proteins of HTNV and DOBV (31 of 36 and one of two in the two groups). In the group of sera selected for serotyping by focus reduction neutralization assay, this dominance was confirmed by the identification of eight DOBV but only four PUUV infections. No infection by HTNV or another hantavirus besides DOBV and PUUV was verified. Anti-hantavirus-positive human sera were detected in all seven investigated counties of Lithuania. CONCLUSION: In Lithuania at least two hantaviruses, DOBV and PUUV, circulate and cause human infections. Additional investigations are needed to study the seroprevalence more precisely and to search for clinical cases of hantavirus infections.

Comparison of cell culture-grown JC virus (primary human fetal glial cells and the JCI cell line) and recombinant JCV VP1 as antigen for the detection of anti-JCV antibody by haemagglutination inhibition.

JC virus (JCV) is the causative agent of the demyelinating disease progressive multifocal leucoencephalopathy (PML), which can be diagnosed by detection in the cerebrospinal fluid (CSF) of both JCV DNA and intrathecally-produced anti-JCV antibody. However, the restricted in-vitro species and cell tropism shown by JCV has made antigen production difficult and limited serological investigations both in PML diagnosis and for JCV epidemiology. In this study antigen prepared as a crude cell lysate of JCV-infected primary human fetal glial (PHFG) cells was compared in a haemagglutination inhibition (HI) assay with antigen produced from the JCV carrier cell line, JCI, and yeast-expressed JCV VP1. Forty-two sera were tested with each antigen and there was a high level of correlation between the assays: 96.5% between the HI assays with PHFG and JCI antigens and 98.1% between the HI assays with PHFG and recombinant VP1 (rVP1) antigens. The JCI antigen gave HI titres 19% lower than the PHFG antigen (P=0.022). Titres with the rVP1 antigen were 2% higher than with the PHFG antigen (P=0.83). When serum/CSF pairs from 11 PML patients were tested, the antibody index calculated in each case confirmed the production of intrathecal anti-JCV antibody. Antibody testing for JCV is no longer reliant on PHFG cells and JCV serological tests should be available more widely.

[Chimeric yeast prions with unstable inheritance]. / Khimernye priony drozhzhei s nestabil'nym nasledovaniem.

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressory effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.

Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.

OBJECTIVES: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

Formation of immunogenic virus-like particles by inserting epitopes into surface-exposed regions of hamster polyomavirus major capsid protein.

We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-S1 region of the hepatitis B virus (designated S1; amino acid sequence DPAFR) was introduced into the predicted positions of VP1. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57Bl6 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3. Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development.

Heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1) using versatile autonomously replicating vector for a wide range of host.

A versatile plasmid shuttle vector system pKDU7 was constructed, which is useful for the heterologous gene expression in a wide range of Kluyveromyces and Saccharomyces strains. This cloning vector was constructed using the 1.6-µm circular plasmid pKD1 of Kluyveromyces drosophilarum, the URA3 gene of K. marxianus as well as the pUC19 sequences. The stability of vector in transformants strongly depends on the integrity of the functionally important elements of pKD1. It was shown by comparison of three recombinant vectors, which possessed the pKD1 sequence inserted in different ways. The efficient transformation and stability maintenance of the vector constructed in various strains of Kluyveromyces and Saccharomyces was shown by the expression of the EPG1 gene of the Kluyveromyces marxianus encoding pectin-degrading endopolygalacturonase.

Use of gene shuffles to study the cytoplasmic transcription system operating on Kluyveromyces lactis linear DNA plasmids.

A modified double selection approach for manipulation of the cytoplasmic plasmids k1 and k2 from dairy yeast Kluyveromyces lactis has been exploited to investigate promoter and gene function. Using TRP1-mediated integration of a LEU2 gene fusion, we have shown that expression of the selection marker is strictly dependent on the k2 promoter UCS5. Also, k2ORF6, the gene encoding the RNA polymerase specific for UCS recognition, is functional when shuffled between the plasmids. Once transplaced onto k1 by means of gene shuffling, the hybrid ORF6 complemented an orf6 deletion created on plasmid k2 eventually yielding yeast strains that contained only two recombinant plasmids: a k2 derivative (rk2/6) with a k2orf6::TRP1 gene deletion, and a k1 derivative (rk1/6) carrying the transplaced ORF6 allele along with the LEU2 marker. This interchangeability of both UCS promoter activity and gene function between k2 and k1 supports the concept of an autonomous transcription system that operates on these nonconventional yeast plasmids.

Yeast cells allow high-level expression and formation of polyomavirus-like particles.

Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1).

The yeast Kluyveromyces marxianus strain BKM Y-719 produces an efficient pectin-degrading endopolygalacturonase (EPG) that cleaves the internal alpha-1,4-D-glycosidic linkages to yield oligomers of varying sizes. The EPG1 gene encoding this industrially important EPG was cloned by using the polymerase chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragment contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed similarity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C218XGGHGXSIGSVG230) that is usually associated with a PG active site. Pulsed-field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y-719 and using chromoblotting it seems that EPG1 is present as only a single copy in the genome.

First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania.

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5' and 3' parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with "universal" Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.

Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae.

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4+ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.

Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae.

A gene (FDH1) of Candida maltosa which confers resistance to formaldehyde in Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The gene has a single intron which possesses the highly conserved splicing signals found in S. cerevisiae introns. We demonstrated that processing of the pre-mRNA of the cloned gene occurred identically in both S. cerevisiae and C. maltosa. The predicted amino acid sequence from the cloned gene showed 65.5% identity to human alcohol dehydrogenase (ADH) class III and 23.9% identity to S. cerevisiae ADH1. The most probable mechanism of resistance to formaldehyde is thought to be the glutathione-dependent oxidation of formaldehyde which is characteristic for ADH class III. The cloned FDH1 gene was successfully employed as a dominant selectable marker in the transformation of S. cerevisiae.

Cloning and sequence analysis of a Candida maltosa gene which confers resistance to cycloheximide.

A CYHR gene from Candida maltosa, which confers resistance to cycloheximide, was cloned in Saccharomyces cerevisiae. A 2.3-kb DNA fragment carrying this gene was sequenced, and an open reading frame able to encode 553 amino acids (aa) was found in the sequence. Computer searches of the GenBank, EMBL, SWIS-PROT and Gen-Pept databases using the FASTA program failed to detect any proteins with extensive similarities to the deduced aa sequence for CYHR. The cloned gene transforms S. cerevisiae at a frequency similar to auxotrophic markers and can be used as a dominant selectable marker for introducing recombinant plasmids into wild-type strains of S. cerevisiae, as well as for gene disruption experiments.

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.