Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.

The r1 and b1 genes of maize, each derived from the chromosomes of two progenitors that hybridized >4.8 million years ago (MYA), have been a rich source for studying transposition, recombination, genomic imprinting, and paramutation. To provide a phylogenetic context to the genetic studies, we sequenced orthologous regions from maize and sorghum (>600 kb) surrounding these genes and compared them with the rice genome. This comparison showed that the homologous regions underwent complete or partial gene deletions, selective retention of orthologous genes, and insertion of nonorthologous genes. Phylogenetic analyses of the r/b genes revealed that the ancestral gene was amplified independently in different grass lineages, that rice experienced an intragenomic gene movement and parallel duplication, that the maize r1 and b1 genes are descendants of two divergent progenitors, and that the two paralogous r genes of sorghum are almost as old as the sorghum lineage. Such sequence mobility also extends to linked genes. The cisZOG genes are characterized by gene amplification in an ancestral grass, parallel duplications and deletions in different grass lineages, and movement to a nonorthologous position in maize. In addition to gene mobility, both maize and rice regions experienced recent transposition (<3 MYA).

It is generally believed that maize (Zea mays L. ssp. mays) arose as a tetraploid; however, the two progenitor genomes cannot be unequivocally traced within the genome of modern maize. We have taken a new approach to investigate the origin of the maize genome. We isolated and sequenced large genomic fragments from the regions surrounding five duplicated loci from the maize genome and their orthologous loci in sorghum, and then we compared these sequences with the orthologous regions in the rice genome. Within the studied segments, we identified 11 genes that were conserved in location, order, and orientation. We performed phylogenetic and distance analyses and examined the patterns of estimated times of divergence for sorghum and maize gene orthologs and also the time of divergence for maize orthologs. Our results support a tetraploid origin of maize. This analysis also indicates contemporaneous divergence of the ancestral sorghum genome and the two maize progenitor genomes about 11.9 million years ago (Mya). On the basis of a putative conversion event detected for one of the genes, tetraploidization must have occurred before 4.8 Mya, and therefore, preceded the major maize genome expansion by gene amplification and retrotransposition.

Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10(-4)-10(-5)) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions.

We cloned the dbl-1 gene, a C. elegans homolog of Drosophila decapentaplegic and vertebrate BMP genes. Loss-of-function mutations in dbl-1 cause markedly reduced body size and defective male copulatory structures. Conversely, dbl-1 overexpression causes markedly increased body size and partly complementary male tail phenotypes, indicating that DBL-1 acts as a dose-dependent regulator of these processes. Evidence from genetic interactions indicates that these effects are mediated by a Smad signaling pathway, for which DBL-1 is a previously unidentified ligand. Our study of the dbl-1 expression pattern suggests a role for neuronal cells in global size regulation as well as male tail patterning.

When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. Here we show that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. We also show that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. Our observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration.

The VP16 protein of herpes simplex virus is a potent transcriptional activator of the viral immediate early genes. The transcriptional activation region of VP16 can be divided into two functional subregions, here designated VP16N (comprising amino acids 413-456) and VP16C (amino acids 450-490). Assays of VP16C mutants resulting from both random and alanine-scanning mutagenesis indicated that the sidechains of three phenylalanines (at positions 473, 475 and 479) and one acidic residue (glutamate 476) are important for transcriptional activation. Aromatic and bulky hydrophobic amino acids were effective substitutes for each of the three Phe residues, whereas replacement with smaller or polar amino acids resulted in loss of transcriptional function. In contrast, many changes were tolerated for Glu476, including bulky hydrophobic and basic amino acids, indicating that the negative charge at this position contributes little to the function of this subregion. Similar relative activities for most of the mutants were observed in yeast and in mammalian cells, indicating that the structural requirements for this activation region are comparable in these two species. These results reinforce the hypothesis that bulky hydrophobic residues, not acidic residues, are most critical for the activity of this 'acidic' transcriptional activation region.

Drosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that beta4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the beta4GalNAcT trisaccharide acceptor structure GlcNAcbeta,3Manbeta,4GlcbetaCer, are lethal. In contrast, flies doubly mutant for the beta4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in beta4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the beta4GalNAcT single mutants we show that beta4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In beta4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in beta4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that beta4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both beta4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.

Mycobacterium tuberculosis is arguably the world's most successful infectious agent because of its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobacteria in culture and during infection of mice. CarD depletion leads to sensitivity to killing by oxidative stress, starvation, and DNA damage, accompanied by failure to reduce rRNA transcription. CarD can functionally replace DksA for stringent control of rRNA transcription, even though CarD associates with a different site on RNA polymerase. These findings highlight a distinct molecular mechanism for regulating rRNA transcription in mycobacteria that is critical for M. tuberculosis pathogenesis.

Homeostasis in the Drosophila midgut is maintained by stem cells [1, 2]. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate [3, 4], and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell [3-5]. If the midgut epithelium is damaged, then ISC proliferation increases [6-12]. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation [7, 9, 13, 14]. We show that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer [15, 16]. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. Our observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration.

The decapentaplegic (dpp) locus of Drosophila melanogaster is a greater than 55 kb genetic unit required for proper pattern formation during the embryonic and imaginal development of the organism. We have proposed that these morphogenetic functions result from the action of a secreted transforming growth factor-beta (TGF-beta)-related protein product encoded by dpp. In this paper we localize 60 mutations on the molecular map of dpp. The positions of these mutations cluster according to phenotypic class, identifying the locations of specific dpp functions. By Northern and cDNA analysis, we characterize five overlapping dpp transcripts. On the basis of the locations of the overlaps relative to a previously sequenced cDNA, it is likely that these transcripts all encode similar or identical polypeptides. We propose that the bulk of dpp DNA consists of extensive arrays of cis-regulatory information. The large (greater than 25-kb) 3' cis-regulatory region represents a novel feature of dpp gene organization

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within error to the resistance rate for Rif (3 × 10(-8) to 10 × 10(-8) per generation). Substitutions conferring Myx resistance were obtained in the RNAP β subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127(1322)P] and the RNAP β' subunit [five substitutions: K334(345)N, T925(917)K, T925(917)R, G1172(1354)C, and G1172(1354)D] (residues numbered as in Staphylococcus aureus RNAP and, in parentheses, as in Escherichia coli RNAP). Sites of substitutions conferring Myx resistance map to the RNAP switch region SW1/SW2 subregion and do not overlap the binding site on RNAP for Rif, and, correspondingly, Myx-resistant mutants exhibit no cross-resistance to Rif. All substitutions conferring Myx resistance exhibit significant fitness costs (4 to 15% per generation). In contrast, at least three substitutions conferring Rif resistance exhibit no fitness costs (≤0% per generation). The observation that all Myx-resistant mutants have significant fitness costs whereas at least three Rif-resistant mutants have no fitness costs, together with the previously established inverse correlation between fitness cost and clinical prevalence, suggests that Myx resistance is likely to have lower clinical prevalence than Rif resistance.

We have isolated and sequenced all 23 members of the 22-kD alpha zein (z1C) gene family of maize. This is one of the largest plant gene families that has been sequenced from a single genetic background and includes the largest contiguous genomic DNA from maize with 346,292 bp to date. Twenty-two of the z1C members are found in a roughly tandem array on chromosome 4S forming a dense gene cluster 168,489-bp long. The twenty-third copy of the gene family is also located on chromosome 4S at a site approximately 20 cM closer to the centromere and appears to be the wild-type allele of the floury-2 (fl2) mutation. On the basis of an analysis of maize cDNA databases, only seven of these genes appear to be expressed including the fl2 allele. The expressed genes in the cluster are interspersed with nonexpressed genes. Interestingly, some of the expressed genes differ in their transcriptional regulation. Gene amplification appears to be in blocks of genes explaining the rapid and compact expansion of the cluster during the evolution of maize.

Functional analysis of chromosomal segments containing linked genes requires the insertion of contiguous genomic sequences from bacterial artificial chromosomes (BACs) into the genome. Therefore, we introduced a 90-kb large BAC clone carrying a 10-copy tandem array of kafirin storage protein genes from sorghum linkage group J, mixed with a selectable marker gene, directly into maize cells using the particle bombardment method. Transgenic plants were regenerated and seeds from eight different transgenic lines were produced. One such transgenic plant was selected that had the entire kafirin gene cluster on a single continuous DNA fragment spanning more than 45 kb integrated into its genome. When alcohol-soluble proteins from individual T2 and T3 seeds of this event were analyzed, significant levels of kafirin were found in addition to the endogenous zein storage proteins, demonstrating that the large exogenous DNA segment is stably integrated into the maize genome and expressed at high levels in subsequent generations. Therefore, we could provide a new utility of plant transformation by the particle bombardment method for functional genomics of multigene families and the modification of the nutritive quality of cereal grains. Despite a tandem array of highly homologous sequences at the transgenic locus, no gene silencing was observed, probably owing to the effects of co-transformed flanking sequences. The expression studies of the transgenic locus also revealed new features of storage protein gene promoters that differed from previous transient gene expression studies, thereby illustrating the significance of the concentration and configuration of DNA-protein interactions in the regulation of gene expression.

Although comparative genetic mapping studies show extensive genome conservation among grasses, recent data provide many exceptions to gene collinearity at the DNA sequence level. Rice, sorghum, and maize are closely related grass species, once sharing a common ancestor. Because they diverged at different times during evolution, they provide an excellent model to investigate sequence divergence. We isolated, sequenced, and compared orthologous regions from two rice subspecies, sorghum, and maize to investigate the nature of their sequence differences. This study represents the most extensive sequence comparison among grasses, including the largest contiguous genomic sequences from sorghum (425 kb) and maize (435 kb) to date. Our results reveal a mosaic organization of the orthologous regions, with conserved sequences interspersed with nonconserved sequences. Gene amplification, gene movement, and retrotransposition account for the majority of the nonconserved sequences. Our analysis also shows that gene amplification is frequently linked with gene movement. Analyzing an additional 2.9 Mb of genomic sequence from rice not only corroborates our observations, but also suggests that a significant portion of grass genomes may consist of paralogous sequences derived from gene amplification. We propose that sequence divergence started from hotspots along chromosomes and expanded by accumulating small-scale genomic changes during evolution.

A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.

Genomic regions of nearly every species diverged into different haplotypes, mostly based on point mutations, small deletions, and insertions that do not affect the collinearity of genes within a species. However, the same genomic interval containing the z1C gene cluster of two inbred lines of Zea mays significantly lost their gene collinearity and also differed in the regulation of each remaining gene set. Furthermore, when inbreds were reciprocally crossed, hybrids exhibited an unexpected shift of expression patterns so that "overdominance" instead of "dominance complementation" of allelic and nonallelic gene expression occurred. The same interval also differed in length (360 vs. 263 kb). Segmental rearrangements led to sequence changes, which were further enhanced by the insertion of different transposable elements. Changes in gene order affected not only z1C genes but also three unrelated genes. However, the orthologous interval between two subspecies of rice (not rice cultivars) was conserved in length and gene order, whereas changes between two maize inbreds were as drastic as changes between maize and sorghum. Given that chromosomes could conceivably consist of intervals of haplotypes that are highly diverged, one could envision endless breeding opportunities because of their linear arrangement along a chromosome and their expression potential in hybrid combinations ("binary" systems). The implication of such a hypothesis for heterosis is discussed.

Polar auxin transport, mediated by the PIN-FORMED (PIN) class of auxin efflux carriers, controls organ initiation in plants. In maize, BARREN INFLORESCENCE2 (BIF2) encodes a serine/threonine protein kinase co-orthologous to PINOID (PID), which regulates the subcellular localization of AtPIN1 in Arabidopsis. We show that BIF2 phosphorylates ZmPIN1a, a maize homolog of AtPIN1, in vitro and regulates ZmPIN1a subcellular localization in vivo, similar to the role of PID in Arabidopsis. In addition, bif2 mutant inflorescences have lower auxin levels later in development. We propose that BIF2 regulates auxin transport through direct regulation of ZmPIN1a during maize inflorescence development.

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.

In addition to quantitative differences in morphogen signaling specifying cell fates, the vector and slope of morphogen gradients influence planar cell polarity (PCP) and growth. The cadherin Fat plays a central role in this process. Fat regulates PCP and growth through distinct downstream pathways, each involving the establishment of molecular polarity within cells. Fat is regulated by the cadherin Dachsous (Ds) and the protein kinase Four-jointed (Fj), which are expressed in gradients in many tissues. Previous studies have implied that Fat is regulated by the vector and slope of these expression gradients. Here, we characterize how cells interpret the Fj gradient. We demonstrate that Fj both promotes the ability of Fat to bind to its ligand Ds and inhibits the ability of Ds to bind Fat. Consequently, the juxtaposition of cells with differing Fj expression results in asymmetric Fat:Ds binding. We also show that the influence of Fj on Fat is a direct consequence of Fat phosphorylation and identify a phosphorylation site important for the stimulation of Fat:Ds binding by Fj. Our results define a molecular mechanism by which a morphogen gradient can drive the polarization of Fat activity to influence PCP and growth.

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.