Properties of a Trifluoroleucine-Resistant Mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

Asp578 in LEU4p is one of the key residues for leucine feedback inhibition release in sake yeast

We identified a new mutation, Asp578Tyr, in alpha-isopropylmalate synthase (a LEU4 gene product) that releases leucine feedback inhibition and causes hyperproduction of isoamyl alcohol (i-AmOH) in sake yeast. Spontaneous sake yeast mutants that express resistance to 5,5,5-trifluoro-DL-leucine (TFL) were isolated, and a mutant strain, TFL20, was characterized at the genetic and biochemical levels. An enzyme assay for alpha-isopropylmalate synthase showed that strain TFL20 was released from feedback inhibition by L-leucine. Furthermore, DNA sequencing of the LEU4 gene for a haploid of the mutant TFL20 revealed that aspartic acid in position 578 changes to tyrosine. A comparison of the three-dimensional structures of wild-type LEU4p and mutant LEU4D578Yp by the homology modeling method showed that Asp578 is important for leucine feedback inhibition. We conclude that the mutation from Asp to Tyr in 578 is a novel change causing release from leucine feedback inhibition.     

Introduction of a feed back resistant α-isopropylmalate synthase gene of Saccharomyces cerevisiae into sake yeast

A mutant LEU4 gene (LEU4^fbr-2), responsible for both the overproduction of iso-amyl alcohol in yeast and the phenotype of yeast resistant to 5,5,5-trifluoro-dl-leucine (TFL), was isolated from a TFL-resistant mutant of Saccharomyces cerevisiae F-7. The single copy number of LEU4^fbr-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, leu4, leu5), and also transformed it to TFL-resistant. Leucine-insensitive α-isopropylmalate synthase activity was detected in the crude extract of the Leu⁺ transformant. Also sake yeast Kyokai no. 7 (K-7) was transformed by the LEU4^fbr-2 gene to TFL-resistant. The resulting transformants produced 3∼30-fold higher levels of iso-amyl alcohol (approx. 50∼475 ppm) in shaking cultures, while in static cultures the increase in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4^fbr-2 gene may be useful as a positive selectable marker for the transformation of industrial yeast.     

The Arabidopsis heterochromatin protein1 homolog (TERMINAL FLOWER2) silences genes within the euchromatic region but not genes positioned in heterochromatin

TERMINAL FLOWER2 (TFL2) is the only homolog of heterochromatin protein1 (HP1) in the Arabidopsis genome. Because proteins of the HP1 family in fission yeast and animals act as key components of gene silencing in heterochromatin by binding to histone H3 methylated on lysine 9 (K9), here we examined whether TFL2 has a similar role in Arabidopsis. Unexpectedly, genes positioned in heterochromatin were not activated in tfl2 mutants. Moreover, the TFL2 protein localized preferentially to euchromatic regions and not to heterochromatic chromocenters, where K9-methylated histone H3 is clustered. Instead, TFL2 acts as a repressor of genes related to plant development, i.e. flowering, floral organ identity, meiosis and seed maturation. Up-regulation of the floral homeotic genes PISTILLATA, APETALA3, AGAMOUS and SEPALLATA3 in tfl2 mutants was independent of LEAFY or APETALA3, known activators of the above genes. In addition, transduced APETALA3 promoter fragments as short as 500 bp were sufficient for TFL2-mediated gene repression. Taken together, TFL2 silences specific genes within euchromatin but not genes positioned in heterochromatin of Arabidopsis.     

Cloning, Disruption and Chromosomal Mapping of Yeast LEU3 , a Putative Regulatory Gene

The LEU3 gene of the yeast Saccharomyces cerevisiae, which is involved in the regulation of at least two LEU structural genes (LEU1 and LEU2), has been cloned by complementation of leu3 mutations and shown to reside within a 5.6-kb fragment. Transformation of leu3 mutants with LEU3-carrying multicopy plasmids restored normal, leucine-independent growth behavior in the recipients. It also restored approximately wild-type levels of isopropylmalate isomerase (LEU1) and beta-isopropylmalate dehydrogenase (LEU2), which were strongly reduced when exogenous leucine was supplied. Strains containing a disrupted leu3 allele were constructed by deleting 0.7-kb of LEU3 DNA and inserting the yeast HIS3 gene in its place. Like other leu3 mutants, these strains were leaky leucine auxotrophs, owing to a basal level of expression of LEU1 and LEU2. Southern transfer and genetic analyses of strains carrying a disrupted leu3 allele demonstrated that the cloned gene was LEU3, as opposed to a suppressor. Disruption of LEU3 was performed also with a diploid and shown to be nonlethal by tetrad analysis. Northern transfer experiments showed that the LEU3 gene produces mRNA approximately 2.9 kilonucleotides in length. The leu3 marker was mapped to chromosome XII by the spo11 method. Linkage to ura4 by about 44 centiMorgans places leu3 on the right arm of this chromosome.     

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.     

Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes

Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.     

Acceleration of flowering by overexpression of MFT (MOTHER OF FT AND TFL1)

MFT (MOTHER OF FT AND TFL1) is a member of a gene family that includes two important regulators, FT (FLOWERING LOCUS T) and TFL1 (TERMINAL FLOWER 1), in determination of flowering time in Arabidopsis. Although the functions of FT and TFL1 are assigned in the family, the roles of other members are largely unknown. Especially the sequence of MFT is homologous to both FT and TFL1, which act as a floral promoter and an inhibitor, respectively, making it difficult to predict the role of MFT. We performed genetic analyses of MFT to understand its role in floral development. Constitutive expression of MFT led to slightly early flowering under long days. However, a T-DNA insertion allele of MFT did not show obvious phenotype. Further genetic analyses with the loss-of-function alleles of FT, TFL1, and ATC (Arabidopsis Thaliana CENTRORADIALIS homologue) showed that a decrease of MFT activity did not enhance the phenotypes of the single mutants. Taken together, we suggest that MFT functions as a floral inducer and that it may act redundantly in determination of flowering time in Arabidopsis.     

Multiple New Genes That Determine Activity for the First Step of Leucine Biosynthesis in Saccharomyces Cerevisiae

The first step in the biosynthesis of leucine is catalyzed by α-isopropylmalate (α-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of α-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu(+) and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of α-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an α-IPM synthase activity. The results demonstrate that multiple components determine the residual α-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.     

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.     

Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product

It has been known that enzyme activity associated with the yeast LEU1 and LEU2 gene product (beta-isopropylmalate dehydrogenase) drops sharply when yeast is grown in the presence of leucine. RNA blot hybridizations with LEU2-specific probes establish that this is accompanied by a 5-fold repression in LEU2 mRNA levels. A similar repression was noted recently for LEU1 mRNA levels (Hsu, Y.-P., and Schimmel, P. (1984) J. Biol. Chem. 259, 3714-3719). Nuclease mapping of the 5'-end of the LEU2 mRNA shows a major start at approximately 16 nucleotides upstream of the AUG initiation codon. This initiation site in the gene is retained in an extensive LEU2 5'-noncoding region deletion which still expresses the LEU2 gene product (Erhart, E., and Hollenberg, C. P. (1983) J. Bacteriol. 156, 625-635). The primary structure of the LEU2 gene product was established from the nucleotide sequence of the gene-coding region and from fitting amino acid sequences of scattered internal peptides to the nucleotide sequence. The 364-amino acid protein has a 13-amino acid stretch which is highly homologous to the partially sequenced yeast LEU1 gene product (isopropylmalate isomerase). The homology occurs about 290 amino acids from the respective NH2 termini of the two proteins. The homology may represent residues which interact with beta-isopropylmalate, a common ligand for the enzymes.     

花序分生组织特性基因TFL1的系统发育及其功能分析

TFL1同源基因在维持植物营养生长和花序分生组织特性方面起着非常重要的作用,其功能的丧失常导致植物提早开花,花序的正常发育受到抑制,最终茎端形成顶花。至今已经有28种植物的TFL1基因被克隆到,其中包括拟南芥、金鱼草和番茄等模式植物。TFL1 蛋白的系统发育树基本符合物种的亲缘关系。作为花序分生组织特性基因的TFL1与花分生组织特性基因LFY 和AP1相互作用,抑制花序分生组织向花分生组织的转变。TFL1和LFY等基因可用来培育早花新品种,也可用于培育无果的新品种,减少悬铃木、杨、柳等果毛的污染。     

Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.