CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinase domain

The cell division cycle gene CDC15 is essential for the late nuclear division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC15 gene product, as deduced from the nucleotide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/threonine kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth are known. CDC15 represents an essential protein kinase like CDC7 and CDC28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.     

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology. The complete nucleotide sequence was submitted to the EMBL Data Library under Accession Number X83690.     

The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase.

The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from [gamma-32P]ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.     

The SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae

Saccharomyces cerevisiae strains carrying snf3 are defective in high affinity glucose transport, and thus are unable to grow fermentatively on media with low concentrations of glucose. A multicopy suppressor of the snf3 growth defect, SKS1 (suppressor kinase of snf3), was found to encode a putative ser/thr protein kinase homologous to Ran1p, a kinase that regulates the switch between meiosis and vegetative growth in Schizosaccharomyces pombe. Overexpression of the SKS1 open reading frame is sufficient for suppression of the growth defects of snf3 mutants. Disruption of the open reading frame eliminates this suppression; as does the mutation of the consensus ATP binding site of Sks1p. A DDSE (DNA dependent snf3 suppressor element) was found to be present in the SKS1 promoter region. The suppression by this DDSE occurs in the absence of SKS1 coding region, that is, the DDSE can suppress a snf3 sks1 double null mutant which fails to grow fermentatively on low glucose as a snf3 mutant does. Both SKS1 and its DDSE can additionally suppress the growth defects of grr1 mutants, which are also impaired in high affinity glucose transport. The snf3 genomic suppressors, rgt1, RGT2 and ssn6, are also capable of suppressing snf3 associated growth defects in a strain lacking sks1.     

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology

Pab1, the major poly (A) binding protein of the yeast Saccharomyces cerevisiae, is involved in many intracellular functions associated with mRNA metabolism, such as mRNA nuclear export, deadenylation, translation initiation and termination. Pab1 consists of four RNA recognition motifs (RRM), a proline-rich domain (P) and a carboxy-terminal (C) domain. Due to its modular structure, Pab1 can simultaneously interact with poly (A) tails and different proteins that regulate mRNA turnover and translation. Furthermore, Pab1 also influences cell physiology under stressful conditions by affecting the formation of quinary assemblies and stress granules, as well as by stabilizing specific mRNAs to allow translation re-initiation after stress. The main goal of this review is to correlate the structural complexity of this protein with the multiplicity of its functions.     

Sequencing of a 40·5 kb Fragment Located on the Left Arm of Chromosome VII from Saccharomyces cerevisiae

The nucleotide sequence of a 40·5 kb DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae was determined and analysed. Twenty-eight open reading frames (ORFs) longer than 300 nucleotides were identified. Eight of them correspond to the following known yeast genes: EMP24, GCN1, SPO8, COX13, CDC55, RPS26, COX4 and LSR1, also called GTS1. Twelve ORFs are new, among them eight show homology with other genes while four have no homology with any sequence in the databases. Eight additional ORFs are internal to or partially overlapping with other ORFs. The nucleotide sequence reported here is deposited in the EMBL database under the Accession Numbers X91837 and X91489. © 1997 by John Wiley & Sons, Ltd.     

The Saccharomyces cerevisiae TGL2 gene encodes a protein with lipolytic activity and can complement an Escherichia coli diacylglycerol kinase disruptant

Escherichia coli cells with a disrupted diacylglycerol kinase gene are unable to grow on media containing arbutin due to a lethal accumulation of diacylglycerol. In order to isolate genes from the yeast Saccharomyces cerevisiae involved in diacylglycerol metabolism we complemented an E. coli diacylglycerol kinase disruptant with a yeast genomic library and transformants were selected capable of growing in the presence of arbutin. Using this method, a gene (TGL2) was isolated coding for a protein resembling lipases from Pseudomonas. After expression of the TGL2 gene in E. coli, lipolytic activity towards triacylglycerols and diacylglycerols with short-chain fatty acids could be measured. Therefore, it is very likely that the TGL2 gene can complement the E. coli diacylglycerol kinase disruptant, because it encodes a protein that degrades the diacylglycerol accumulated after growth in the presence of arbutin. Disruption of the TGL2 gene in S. cerevisiae did not result in a detectable phenotype. The role of the Tgl2 protein in lipid degradation in yeast is still unclear. The nucleotide sequence published here has been submitted to the EMBL sequence data bank and is available under accession number X98000. © 1998 John Wiley & Sons, Ltd.     

Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).     

The complete sequence of a 15 820 bp segment of Saccharomyces cerevisiae chromosome XI contains the UBI2 and MPL1 genes and three new open reading frames

As part of the EEC yeast genome program, a fragment of 15 820 bp from the right arm of Saccharomyces cerevisiae chromosome XI has been sequenced. This fragment corresponds roughly to the centromere-distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as the UBI2 gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as the MPL1 gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso1.     

The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames.

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MAT alpha locus. The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.     

Cost‐effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins,including phosphorylated eIF2α

The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low‐abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost‐effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short‐term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd.     

The sequence of 36·8 kb from the left arm of chromosome XIV reveals 24 complete open reading frames: 18 correspond to new genes,one of which encodes a protein similar to the human myotonic dystrophy kinase

We have determined the complete nucleotide sequence of a 36·8 kb segment from the left arm of chromosome XIV carried by the cosmid 14–11. The sequence encodes the 5′ coding region of the PSD1 gene, the 3′ coding region of an unknown gene and 24 complete open reading frames, of which 18 correspond to new genes and six (SKO1, SCL41A, YGP1, YCK2, RPC31 and MFA2) have been sequenced previously. Of the 24 new genes, five show significant similarities to sequences present in the databanks. These include elongation factors 2 and the human myotonic dystrophy kinase. The sequence has been deposited in the EMBL databank under the Accession Number X92517.     

XIV. Yeast sequencing reports. Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV carrying a ribosomal protein gene cluster,the genes encoding a plasma membrane protein and a subunit of replication factor C,and a novel putative serine/threonine protein kinase gene

We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases. The sequence has been submitted to the EMBL data library under Accession Number U23084.     

Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10.7 kb. The sequence was determined using a double-strand sequencing strategy adapted from the random-clone strategy. The segment contains seven non-overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumably a protein kinase.     

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames,including a homologue of the KIN1/KIN2 and SNF1 protein kinases

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5′ part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.     

Analysis of a 42·5 kb DNA sequence of chromosome X reveals three tRNA genes and 14 new open reading frames including a gene most probably belonging to the family of ubiquitin–protein ligases

We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8. This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids. Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms. It may belong to the family of ubiquitin–protein ligases and be involved in the ubiquitin-dependent proteolytic pathway. In addition, three tRNA genes were recognized, two of which had not been hitherto localized. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L36344.