Fred Sherman - US grants

Iso-1-cytochrome c and iso-2-cytochrome c from the yeast Saccharomyces cerevisiae are two of the few proteins of known primary structure from a microorganism which is particularly suitable for experimental genetic studies and for manipulation by recombinant DNA procedures. The isolation of appropriate mutants have been facilitated by enrichment procedures for both forward and reverse mutations. A series of deletions are available, making it possible to conveniently map point mutants and to estimate their positions relative to the iso-1-cytochrome c sequence. The large number of mutants that have been characterized and the selection of procedures permit an unprecedented degree of genetic manipulation of nucleotide sequences by recombination. The DNA sequences of the structural genes of iso-1-cytochrome c (CYC1) and iso-2-cytochrome c (CYC7) as well as portions of the adjacent regions have been determined. Thus, the body of information concerning the iso-1-cytochrome c gene, the large number of defined mutants and the available genetic and biochemical techniques are without parallel for any other eukaryotic gene. This iso-cytochrome c system is being employed for investigating numerous problems in molecular biology and genetics, including the following: DNA sequencing, transcriptional analysis, gene assignment and evolutionary relationship of the two 6 kb regions, COR and ARC, which encompass, respectively, the CYC1 and CYC7 genes; regulation and maturation of the two iso-cytochromes c and the role of heme, catabolite repression and anaerobiosis on the synthesis of CYC1 and CYC7 mRNAs and their corresponding apo- and holo-proteins; DNA alterations of the regions adjacent to the CYC1 locus and their effect on initiation and termination of transcription and initiation of translation; investigation of the overproduction of iso-2-cytochrome c caused by alterations in the CYC7 prefix region and by unlinked mutations; DNA sequencing of missense mutations and structure-function relationship of iso-1-cytochrome c; amino acid inserted by ribosomal suppressors and the efficiencies of suppression; relationship of recombination frequencies and nucleotide alterations; investigation of the action of mutagens and the nucleotide sequences of mutable and immutable sites and of unstable mutations.

We wish to establish a state-of-the-art Analytical Protein Microchemistry Facility. This will consist of a gas-phase sequenator and its attendant HPLC amino acid analyzer, an HPLC for protein and peptide purification, and peptide synthesis equipment. With the gas-phase sequenator, automated amino acid sequence analysis can be performed with very small amounts of material (less than 100 picomoles). For a typical protein this corresponds to 5-10 micrograms. These amounts are being produced by monoclonal antibody affinity chromatography or preparative two-dimensional gel electrophoresis in various laboratories of the user group. The proposed equipment will complement existing technical resources at the University including an Oligonucleotide Synthesizer, Cell Hybridoma Lab and Transmission Electron Microscope Facilities. It will serve active research programs in DNA and cDNA cloning, site directed mutagenesis, regulation of gene expression, and structural and functional characterization of proteins. In addition to the individual applications of each machine, a particular feature of these facilities is that they can be used effectively in conjunction. As examples, partial amino acid sequences will serve to define the possible sequences of an oligonucleotide hybridization probe, and these probes used in turn to isolate the genes encoding the initial protein. Conversely, from a DNA sequence one can unambiguously deduce the amino acid sequence. Peptides corresponding to parts of this sequence can be constructed, coupled to a carrier protein, and then used to generate antisera or monoclonal antibodies. These antibodies can be employed to characterize functionally the proteins by stimulating or blocking known functions. Finally, novel gene products may be identified using antibodies directed against peptides synthesized to correspond to open reading frames found by DNA sequence analysis. We envision these technologies complementing one another and the existing expertise in molecular biology, biochemistry, immunology, and tumor biology. They will allow a large number of investigators to address many fundamental questions of clinical and basic science.

This project is in the Chemistry of Life Processes Initiative (COLP) and supported jointly by Chemistry and Biophysics. Fundamental studies of protein structure-function relationships will be carried out. Iso-cytochrome c from yeast is used to study how amino acid replacements and deletions affect key parameters such as electron transfer rates, binding strength, specificity to protein partners of cytochrome c, and protein stability. Detailed intracellelar measurements will be made for comparison with the in vitro work, using growth in lactate medium as a measure of relative specific activity. Specific mutations have been generated for each of the studies. Along with the experimental work, a variety of theoretical approaches are being developed to aid in the design and interpretation of experiments. This project is a continuation of that previously supported under the COLP Initiative.

The two nuclear genes, CYC1 and CYC7, encoding the mitochondrial proteins iso-1- and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae, comprise one of the most thoroughly studied gene-protein systems of eukaryotes. All steps of CYC1 gene expression, have been systematically examined, and methods have been developed for the detection and selection of mutants, for determining the levels of cytochrome c in vivo, and for altering the CYC1 and CYC7 genes by transforming yeast directly with synthetic oligonucleotides. We plan to carryout studies on the following: (1) the Sut1p RNA degradation system; (2) the degradation of cytochrome c, both apo and holo forms; and (3) amino-terminal processing of proteins, including the action of methionine aminopeptidases and amino-terminal acetyltransferases. (1) We will test the working hypothesis that the recently identified Sut1p degradation system is responsible for degrading RNA in nuclei, including abnormal cyc1-512 mRNAs, normal mRNAs retained in nuclei, introns of mRNA, introns of tRNA, and spacer sequences processed from rRNA. Other mutants phenotypically similar to sut1 mutants will be isolated and characterized. The enzymatic activity and other components associated with Sut1p will be investigated, using a GST-SUT1 fused gene, the two hybrid system, and mutants obtained by the synthetic lethality procedure. (2) There appears to be at least four pathways for degrading the holo or apo forms of the two iso-cytochromes c, including degradation by the ubiquitin-dependent pathway. We will investigate the mechanisms by which degradation occurs for: apo-1, but not apo-2; holo having amino-terminal amphipathic structures; apo in the absence of heme in hem1 strains; and altered forms of holo, which are dependent or independent on the cytochromes c1 and a.a3. (3) We will identify and investigate the pattern of action of different methionine aminopeptidases and different amino-terminal acetyltransferases, some of which were uncovered on the basis of sequence similarities. A functional GST-NAT2 fused gene will be used to investigate possible other components required for an amino-terminal acetyltransferase acting on a subset of proteins having methionine termini. We will investigate the proteins from mutants with disrupted genes that normally encode different amino-terminal acetylases or methionine aminopeptidases. The proteins that exhibit altered mobilities on 2-D gels will be identified and sequenced by mass spectrometry.

DESCRIPTION (Adapted from applicant's abstract): Dr. Sherman and his group have recently demonstrated that standard laboratory strains of Candida albicans spontaneously gave rise to different types of colonial morphological mutants that are associated with single and multiple gross chromosomal rearrangements. Some of the mutants were unstable and gave rise to additional colonial forms after further subcloning. More recently, they have shown that the electrophoretic karyotypes of four independent clinical isolates all differed from each other, and some of the chromosomal differences superficially resembled the alterations uncovered in the morphological mutants. They suggest that the chromosomal aberrations arising from normal strains provide a means for genetic variation in this asexual microorganism. This proposal deals with a systematic investigation of the patterns of electrophoretic karyotypes and chromosomal rearrangements associated with morphological mutants and comparisons of these mutants to a variety of clinical isolates. High resolution procedures of pulse field electrophoresis to separate the closely running chromosomes and to detect slight variations of abnormal lengths will be carried out with orthogonal-field alteration-gel electrophoresis (OFAGE) and contour-clamped homogeneous-electric-field gel electrophoresis (CHEF). The chromosomal rearrangements will be characterized by hybridizing separated NotI and SfiI restriction fragments to probes corresponding to sites distributed along each chromosome. The number of rDNA repeats, that could be responsible for the variation of chromosome VIII length observed in the four clinical isolates and in many morphological mutants, will be estimated simply by digesting total DNA with BamHI (or another restriction endonuclease that does not cleave rDNA), separating the fragments with OFAGE or CHEF, and hybridizing blots with a rDNA probe. The results of the comparisons of the mutants and independent clinical isolates may provide credence to the hypothesis that the chromosomal aberrations are providing genetic variation that allows C. albicans to adapt to new conditions of their host. The patterns and types of chromosomal aberrations in the stable and unstable mutants my shed light on mechanisms by which they arise.

Protein synthesis occurs with a high degree of precision that depends on numerous components, such as ribosomal proteins, and standard and novel translation factors. The study of omnipotent suppression in the yeast Saccharomyces cerevisiae is one of the most fruitful approaches for identifying factors involved in translational ambiguity and for uncovering new components of the translational machinery of eukaryotic cells. Furthermore, the yeast S. cerevisiae represents an especially convenient model organism for investigating the translational apparatus of eukaryotes because the genes identified in yeast are usually evolutionarily conserved and have their counterparts in other eukaryotic organisms. Also, the powerful genetic and DNA recombinant methods available for yeast allows convenient disruption and modification of genes. The main aim of the proposed research project is to investigate the functions of proteins encoded by omnipotent suppressors, with an emphasis on SUP45 and SUP35, and to define their roles in the translational process. the Sup45 and Sup35 proteins will be purified and used in yeast cell-free translational systems and for studies of their interactions with aminoacyl- tRNA, GTP and mRNA. Mutations, deletions and oligonucleotide-directed mutagenesis will be used to study the structure-function relationship of these proteins. The specific role in the translational process and their interaction with other components of the translational apparatus will be investigated with cell-free systems. Studies of proteins interacting with the Sup45 and Sup35 proteins will be used as an attempt to define the interrelationships of translation with other processes in yeast. Because the SUP35 and SUP45 genes appear to affect different cellular processes including, respiration and cell cycle control, we plan to study the nuclear localization of the Sup35 and Sup45 proteins by cellular fractionation and by the method of indirect immunofluorescence, and to study of possible interaction of these proteins with DNA. The wild-type genes corresponding to the newly isolated SUP42 and SUP43, and possibly other omnipotent suppressors, will be sequenced. If they appear to be novel translational factors, their function will be further investigated by the approaches described above. Plasmid-mediated amplification of the wild-type genes, leading to omnipotent suppression, will be used as an approach for identification of novel components of translational apparatus involved in maintaining the translational accuracy in eukaryotes.

The aim of the proposed project is to investigate the mitochondrial protein degradation system - a largely neglected system involved in the biogenesis and maintenance of mitochondria and mitochondrial components. We will investigate the mitochondrial protein degrading system in yeast - the organism most frequently used in studies of mitochondrial biogenesis. ATP-dependent protease, a major component of the mitochondrial protein degrading machinery, will be for the first time isolated from yeast mitochondria. The enzyme will be characterized and compared with its mammalian counterpart and with bacterial protease La. Antibodies against the enzyme will be raised and used to determine the enzyme levels under the conditions affecting mitochondrial biogenesis. N-terminus of the enzyme will be microsequenced with the aim to clone the corresponding gene. Hydrolysis of normal and mutationally altered forms of yeast cytochrome c will be compared to the corresponding half-lives in vivo. Possible heat-shock character of the enzyme will be tested and its functional relationship with mitochondrial chaperonins will be examined. ATP-independent protease(s) of yeast mitochondria will be also purified and characterized. The level and activities of the yeast mitochondrial ATP-dependent protease will be examined in yeast under conditions that affect mitochondrial biogenesis, such as catabolite repression and anaerobic grow. A functional link between the mitochondrial respiratory enzymes and the ATP-dependent protease will be investigated. These results should shed light on the hypothesis that the mitochondrial degradation system regulates steady-state level of normal mitochondrial proteins, as well as eliminates abnormally unstable forms.

We are requesting funds for the acquisition of equipment and for renovation of a fermentor to expand and upgrade the Fermentation Facility at the University of Rochester. Although the Fermentation Facility has been in operation for over 25 years, some of the equipment has deteriorated, making it difficult and inefficient to prepare large quantities of yeast and E. coli. The requested new equipment and the renovation will rectify this deficiency at the University of Rochester. The following items are requested for the Fermentation Facility: (i) a BioFlo IV 20 liter fermentor (New Brunswick Scientific Co., Inc.); (ii) the renovation of a 130 liter fermentor that is currently in need of repair; (iii) a Model Z41 continuous flow centrifuge (New Brunswick Scientific Co., Inc.); (iv) a Model 58750 sterilizer (VWR Scientific); and (v) a Labconco Laboratory Washer (VWR Scientific). The BioFlo IV 20 liter fermentor will partially replace and supplement three 16 liter fermentors, purchased in 1983, that are in constant need of repair and are inefficient to operate. The 130 liter fermentor, purchased in 1976 (Model 1401, Fermentation Design), will be renotated by the New Brunswick Scientific Co. The continuous flow centrifuge is required for collection of cells. The autoclave will replace a older model, purchased in 1969, that barely functions. The Laboratory Washer will be used for general purposes, including washing of glassware used for the preparation of precultures. The total cost of the equipment for the Fermentation Facility is $152,667; we are requesting $100,000 from NSF, and the University of Rochester will be provide remaining $52,667. The Fermentation Facility will be used by at least five members of the faculty from the University of Rochester, Drs. F. Sherman, E. Phizicky, T. Platt, A. E. Senior (Department of Biochemistry), and J. S. Butler (Department of Microbiology & Immunology); and by Dr. G. Mclendon, (Department of Chemistry, Universi ty of Rochester, and Department of Chemistry, Princeton University). In addition, the Fermentation Facility will be available to others at the University of Rochester. The Fermentation Facility is housed on the sixth floor of the Medical School (room 6-5721). The Fermentation Facility will be under the responsibility of Dr. F. Sherman and will be managed by Ms. Linda Comfort, who is Laboratory technician working in Dr. Sherman's group. Each research group will be responsible for operating the equipment for their own preparations; however, if necessary, the different groups can consult with technicians having expertise in the use of the equipment. The Fermentation Facility will be primarily used to prepare large quantities of normal and mutant forms of yeast and Escherichia coli strains for a wide range of studies carried out at the University of Rochester. The following investigators require the Fermentation Facility to carry out the following studies: F. Sherman, amino-terminal acetylation and post-translational processing of proteins from yeast; G. Mclendon and F. Sherman, X-ray structures and physical studies of mutant forms of cytochrome c of yeast; E. Phizicky, characterization of components of the tRNA splicing reaction of yeast; T. Platt, identification and characterization of proteins involved in 3'-end formation of yeast mRNA; J. S. Butler, identification and characterization of proteins involved in cleavage and polyadenylation of yeast mRNA; and A. E. Senior, structure-function relationships of the FlFo-ATP synthase from E. coli. All of these studies are funded by either NIH or NSF grants, and all are parts of projects involving graduate and post-doctoral students. In addition, the Fermentation Facility will provide training for undergraduate students, who are hired to operate the fermentors.

DESCRIPTION (Verbatim from Applicant's Abstract): Clinical isolates of the pathogenic yeast Candida albicans exhibit extensive variation in electrophoretic karyotypes and in phenotypic polymorphism. In this connection, systematic studies conducted in our laboratory revealed that laboratory strains of C. albicans spontaneously give rise to high frequencies of many different types of mutants having altered phenotypes and karyotypes. The significance of the chromosomal alterations was established with spontaneous mutants that acquired the ability to utilize alternative carbon sources. A causal relationship was established with a series of Sou- to Sou+ to Sou- to Sou+ derivatives, in which the Sou- (L-sorbose none-utilizing) and Sou+ (L-sorbose utilizing) strains were, respectively, disomic and monosomic for chromosome 5. Furthermore, transcription of the SOU1 gene, required for L-sorbose utilization, was regulated by the copy number of chromosome 5, in spite of the fact that SOU1 resides on a different chromosome. A hypothetical negative regulator, CSU51, was postulated to reside on chromosome 5, such that transcription of SOU1 is dependent on the ratio of the CSU51 to SOU1 copy number. Other examples of negative regulation by chromosome copy number include the utilization of D-arabinose, Aru- to Aru+, and resistance to the antifungal agent, fluconazole, FluS to FluR, thus establishing a general regulatory mechanism. The major long-term goal of the proposed research is to determine mechanisms of this newly-discovered regulatory process in C. albicans, by which gene expression is controlled by chromosome copy number. Several candidates of the negative regulator residing on chromosome 5 have been isolated from a library of chromosome 5 DNA, and these are being characterized. These regulators will be investigated for their direct or indirect interaction with the SOU1 structural gene. The additional negative regulators, which were retrieved from a total genomic library, and which are located on different chromosomes, will be analyzed for their involvement in the regulatory network controlled by chromosome copy number. This work establishes for the first time a negative regulatory network for a secondary carbon source in an important pathogen.

A human gene probably responsible for Batten s disease has recently been cloned. This is a very serious disease causing neurodegeneration and death in many children. The nature of the defect causing the disease is not known. This human gene, CLN3, has a homologue in yeast, BTN1. A btn1 null mutant is viable, but does have a subtle phenotype in that under some conditions the mutant is somewhat resistant to D-(-)three-2-amino-1-[p-nitropheny1]-1,3-propanediol (ANP). The investigators propose to investigate Batten s disease using yeast and the BTN1 gene as a model system. The P.I. s will investigate expression of normal and mutationally altered forms of Cln3p and Btnip, and will determine critical regions of the proteins. The subcellular location of Btnip will be determined using GFP or epitope tags. Proteins interacting with Btnip will be found using the two-hybrid system, as well as by isolating possible complexes using a GST-Ttnip fusion or Btnip tagged with poly-histidine. Genetic screens will be done to find other mutants resistant to APN, to find suppressors of btn1, and to find mutations that are synthetically lethal with btn1.

DESCRIPTION (provided by applicant): We propose to carryout studies of N-terminal acetylation, mRNA degradation, and protein degradation in Saccharomyces cerevisiae. Although conceptually unrelated, these proposed studies stem from investigations with the iso-cytochromes c system, which constitutes one of the most thoroughly studied gene-protein systems of eukaryotes. Yeast contains three N-terminal acetyltransferases (NATs), designated NatA, NatB and NatC, with each having a different catalytic subunit, Ardip, Nat3p and Mak3p, respectively, and each acetylating different sets of proteins with different N-terminal regions. We propose to identify and characterize all of the subunits of NatB and NatA that co-purify with the catalytic subunits, similar to our work on NatC. The N-terminal acetylation patterns will be determined with mutants deleted in other putative NATs. Biological functions in vivo of acetylation of actin, a NatB substrate, will be deduced from the phenotypes of certain act] mutants. Attempt will be made to characterize mammalian orthologues of the yeast NatA. We will determine if NAT components associate with polysomes and which subunits are responsible for the association. We will test the hypothesis that mRNAs retained in the nucleus are degraded (the DRN pathway), that nuclear mRNA is delivered to the site of degradation by the nuclear cap binding complex containing Cbclp, and that Rrp6p carries out this degradation. DNA microarray technology will be used to identify wild-type mRNAs that are particularly susceptible DRN. Components of the DRN pathway will be identified by suppression of cyc1l-512, which produces abnormally long mRNAs that are highly susceptible to degradation by this pathway. Further studies will carried out with novel protein degradation systems uncovered with the isocytochromes c system. Included are those in which mutationally altered holo-iso-1 having T78I and other replacements are degraded (the RDD pathway). Mutationally altered apo-iso-1 having N-terminal amphipathic structures are degraded independent of ubiquitin system (the MDD pathway); and apo-iso-1 is degraded in the absence of heme and independent of ubiquitin system (the HDD pathway). The latter study would provide the first evidence that apo-cytochrome c and heme interact in vivo in the absence of heme lyase.