Sequence and analysis of the tomato JOINTLESS locus.

A 119-kb bacterial artificial chromosome from the JOINTLESS locus on the tomato (Lycopersicon esculentum) chromosome 11 contained 15 putative genes. Repetitive sequences in this region include one copia-like LTR retrotransposon, 13 simple sequence repeats, three copies of a novel type III foldback transposon, and four putative short DNA repeats. Database searches showed that the foldback transposon and the short DNA repeats seemed to be associated preferably with genes. The predicted tomato genes were compared with the complete Arabidopsis genome. Eleven out of 15 tomato open reading frames were found to be colinear with segments on five Arabidopsis bacterial artificial chromosome/P1-derived artificial chromosome clones. The synteny patterns, however, did not reveal duplicated segments in Arabidopsis, where over half of the genome is duplicated. Our analysis indicated that the microsynteny between the tomato and Arabidopsis genomes was still conserved at a very small scale but was complicated by the large number of gene families in the Arabidopsis genome.

Regulation of gene expression by small molecules in rice.

A system for the regulation of gene expression by small molecules in transgenic rice was developed. This gene switch system consists of two components: (1) a hybrid chemically activated transcription factor, and (2) a synthetic target promoter. The two elements were transformed into rice suspension cells and transgenic plants were regenerated. A luciferase reporter under control of the gene switch system displayed as high as 10,000-fold inducibility following exposure to the small molecule ligand. The dose-response and induction time-course were determined. Regulated luciferase activity in activated plants decreased one day following removal of ligand and could be reactivated multiple times without apparent cosuppression. Analysis of luciferase activity following ligand application to media surrounding the roots suggests that ligand can be absorbed and transported systemically. In contrast, reporter activation was limited to a small area when ligand was applied directly to the leaf surface. The described gene switch system represents an important tool for situations requiring conditional gene expression in a monocot species.

Isoform-specific localization of A-RAF in mitochondria.

RAF kinase is a family of isoforms including A-RAF, B-RAF, and C-RAF. Despite the important role of RAF in cell growth and proliferation, little evidence exists for isoform-specific function of RAF family members. Using Western analysis and immunogold labeling, A-RAF was selectively localized in highly purified rat liver mitochondria. Two novel human proteins, which interact specifically with A-RAF, were identified, and the full-length sequences are reported. These proteins, referred to as hTOM and hTIM, are similar to components of mitochondrial outer and inner membrane protein-import receptors from lower organisms, implicating their involvement in the mitochondrial transport of A-RAF. hTOM contains multiple tetratricopeptide repeat (TPR) domains, which function in protein-protein interactions. TPR domains are frequently present in proteins involved in cellular transport systems. In contrast, protein 14-3-3, an abundant cytosolic protein that participates in many facets of signal transduction, was found to interact with C-RAF but not with A-RAF N-terminal domain. This information is discussed in view of the important role of mitochondria in cellular functions involving energy balance, proliferation, and apoptosis and the potential role of A-RAF in regulating these systems.

Rice as a model for cereal genomics.

Over the past two years, selected regions of the rice genome have been sequenced and shown to be colinear at the sequence level with limited regions of other cereal genomes. A large number of expressed gene sequences and molecular markers have accumulated in the public databases. Large insert clone libraries of the rice genome have been constructed, and rice has become an increasingly attractive candidate for whole genome sequencing.

Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

Plant activating sequences: positively charged peptides are functional as transcriptional activation domains.

Plant sequences that act as transcriptional activation domains in yeast as well as in plants have been isolated by genetic selection in yeast. The selection was based on the reconstitution of a functional GAL4 transcriptional activator. Since the peptides show no homology with reported activation domains, they represent a new class of activating sequences. The sequence P1, which is 10 amino acids long, is the shortest functional activation domain reported. A cDNA that encodes the P14 class (peptides P14-P18) activating sequence have been cloned. The protein exhibits strong homology (higher than 50% amino acid identity) with the BBC1-related sequences, a highly conserved family of basic proteins containing nuclear localization signals. The P14 and P15 peptides are the most effective plant activating sequences. The P14 and P15 peptides are highly hydrophilic, positively charged and mostly unstructured. These properties are at odds with the ones usually found in known activation domains.

Ubiquitin pools, ubiquitin mRNA levels, and ubiquitin-mediated proteolysis in aging human fibroblasts.

Senescent cells have less free ubiquitin and more ubiquitin-protein conjugates than do young cells. The ubiquitin-protein conjugates are heterogeneous in size but contain prominent bands at 106, 55, and 22 kDa. The age-related increase in ubiquitin-protein conjugates applies primarily to the 55-kDa band, while the 106-kDa and 22-kDa conjugates change little with age. Ubiquitin mRNA levels do not change with age, and the ability of cells to degrade two proteins that are good substrates for ubiquitin-mediated proteolysis is unaltered by aging. These results indicate that an increase in ubiquitin-protein conjugates does not necessarily reflect alterations in ubiquitin-mediated proteolysis. Furthermore, an overactive pathway of ubiquitin-mediated proteolysis does not appear to contribute to the proliferative arrest in senescent cells.

Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins.

The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.

C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites.

Tissue-specific expression of the maize anthocyanin Bronze-1 (Bz1) gene is controlled by the products of several regulatory genes. These include C1 or Pl and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. Bz1 expression in embryo tissues is dependent on C1 and an R-sc allele of R. Transient expression from mutated and deleted versions of the Bz1 promoter fused to a luciferase reporter gene was measured in C1, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for Bz1 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these "myb" and "myc" sequences reduced Bz1 expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35S minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for Bz1 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C1 and R interact with the Bz1 promoter at these sites.

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

Genes encoding fusions between the maize regulatory protein C1 and the yeast transcriptional activator GAL4 and mutant C1 proteins were assayed for their ability to trans-activate anthocyanin biosynthetic genes in intact maize tissues. The putative DNA-binding region of C1 fused to the transcriptional activation domain of GAL4 activated transcription of anthocyanin structural gene promoters in c1 aleurones, c1 Rscm2 embryos, and c1 r embryogenic callus. Cells receiving these constructs accumulated purple anthocyanin pigments. The C1 acidic region fused to the GAL4 DNA-binding domain activated transcription of a GAL4-regulated promoter. An internal deletion of C1 also induced pigmentation; however, frameshifts in either the amino-terminal basic or carboxy-terminal acidic region blocked trans-activation, and the latter generated a dominant inhibitory protein. Fusion constructs between the wild-type C1 cDNA and the dominant inhibitor allele C1-I cDNA were used to identify the amino acid changes in C1 responsible for the C1-I inhibitory phenotype. Results from these studies establish that amino acids within the myb-homologous domain are critical for transcriptional activation.

Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues.

The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins.

Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La.

To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response.

Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides.

Site-directed mutagenesis using oligonucleotides that are degenerate at a specific codon was employed to construct a set of mutations in a pentapeptide sequence targeting cytosolic proteins to lysosomes during serum withdrawal. Low-temperature annealing of the mixed oligonucleotides to single-stranded phage DNA and a genetic selection for the DNA strand carrying the mutations were utilized. The use of mixed oligonucleotides by this technique provides an economical means of generating a large set of substitution mutations. A single codon can be changed to codons for most other amino acids in one step. This approach eliminates the need for restriction enzyme cleavage sites flanking the target for mutagenesis and, therefore, is useful for targeting mutations to any DNA fragment cloned into an appropriate single-stranded bacteriophage.

An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli.

The lon gene product in Escherichia coli is an ATP-dependent protease (La) that plays an important role in the breakdown of abnormal proteins and certain normal polypeptides. Since transcription of the lon gene rises as part of the heat-shock response, we studied the physiological effects of increased levels of protease La. In cells carrying additional copies of the lon gene under the control of the lac or tac promoter, induction of the protease resulted in a rapid cessation of cell growth and in a loss of viability at stationary phase. Similarly, cells carrying a multicopy plasmid encoding the lon gene contained 2-5-fold more protease La and grew much more slowly than did control cells. In such cells, insertion sequences appeared spontaneously in the lon gene on the plasmid and prevented the excess protease production and allowed more rapid growth. The cells with increased content of protease La (due to the lon plasmid or induction of the lon gene) exhibited severalfold higher rates of degradation of abnormal proteins containing amino acid analogs and of incomplete polypeptides containing puromycin. Also, a beta-galactosidase fusion protein with enzymatic activity was relatively stable in control cells but unstable in the cells with high protease La content. In these cells, the overall degradation of normal proteins increased 2-fold, and certain cellular polypeptides appeared particularly sensitive to proteolysis. Thus, rates of protein degradation in vivo are limited in part by the cellular content of the ATP-dependent protease, and increases in transcription of the lon gene enhance proteolysis and can be deleterious to the cell.

Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes.

The product of the lon gene in Escherichia coli, protease La, plays an important role in the degradation of abnormal proteins. To determine whether the presence of abnormal proteins stimulates expression of this gene, we examined its transcription using a lon-lacZ operon fusion. After the cells synthesized large amounts of aberrant polypeptides (e.g. following incorporation of the arginine analog, canavanine, or production of incomplete proteins with puromycin, or induction of translational errors with streptomycin), these cells showed a two- to threefold increase in lon--lacZ expression. Furthermore, synthesis of a single cloned protein, e.g. human tissue plasminogen activator, caused a similar increase in lon transcription. This induction of lon by abnormal proteins requires the heat shock regulatory gene htpR and was not seen in htpR- cells. Under these various conditions, other heat shock proteins were also induced. Thus, the appearance of aberrant cell proteins may be a common signal under many adverse conditions for the induction of cell protease (or proteases) and other heat shock proteins.

Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli.

Upon a shift to high temperature, Escherichia coli increase their rate of protein degradation and also the expression of a set of "heat shock" genes. Nonsense mutants of htpR (also called hin), suppressed by a temperature-sensitive suppressor, show lower expression of heat shock genes at 30 degrees C and fail to respond to a shift to 42 degrees C. These mutants were found to have a lower capacity to degrade abnormal or incomplete proteins than that of wild-type cells. This reduction in proteolysis equals or exceeds that in lon mutants, which encode a defective ATP-dependent protease, protease La, and is particularly large in htpR lon double mutants. The activity of protease La was higher in wild-type cells than in htpR mutants grown at 30 degrees C and increased upon shift to 42 degrees C only in the wild type. To determine whether htpR influences transcription of the lon gene, a lon-lacZ operon fusion was utilized. Introduction of the htpR mutation reduced transcription from the lon promoter at 30 degrees C and 37 degrees C. This defect was corrected by a plasmid (pFN97) carrying the wild-type htpR allele. Induction of the heat shock response with ethanol had little or no effect in htpR mutants but stimulated lon transcription 2-3 fold in wild-type cells and htpR cells carrying pFN97. Thus, lon appears to be a heat shock gene, and increased synthesis of protease La under stressful conditions may help to prevent the accumulation of damaged cellular protein.