We have isolated two alleles of a previously unidentified meiotic recombination gene, mei-217. Genetic analysis of these mutants shows that mei-217 is a typical "precondition" gene. The phenotypes of the mutants are meiosis specific. The strongest allele has 10% of the normal level of crossing over, and the residual events are distributed abnormally. We have used double mutant analysis to position mei-217 in the meiotic recombination pathway. In general, mutations causing defects in the initiation of meiotic recombination are epistatic to mutations in mei-41 and spnB. These two mutations, however, are epistatic to mei-217, suggesting that recombination is initiated normally in mei-217 mutants. It is likely that mei-217 mutants are able to make Holliday junction intermediates but are defective in the production of crossovers. These phenotypes are most similar to mutants of the mei-218 gene. This is striking because mei-217 and mei-218 are part of the same transcription unit and are most likely produced from a dicistronic message.

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Protocols developed for plastome engineering in Nicotiana tabacum rely on biolistic delivery of the transforming DNA to chloroplasts in intact leaf tissue; integration of the foreign DNA into the plastid genome by homologous recombination via flanking plastid DNA (ptDNA) targeting regions; and gradual dilution of non-transformed ptDNA during cultivation in vitro. Plastid transformation in Arabidopsis was obtained by combining the tobacco leaf transformation protocol with Arabidopsis-specific tissue culture and plant regeneration protocols. Because the leaf cells in Arabidopsis are polyploid, this protocol yielded sterile plants. Meristematic cells in a shoot apex or cells of a developing embryo are diploid. Therefore, we developed a regulated embryogenic root culture system that will generate diploid tissue for plastid transformation. This embryogenic culture system is created by steroid-inducible expression of the BABY BOOM transcription factor. Plastid transformation in Arabidopsis will enable the probing of plastid gene function, and the characterization of posttranscriptional mechanisms of gene regulation and the regulatory interactions of plastid and nuclear genes.

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1(v) gene) or a synthetic sequence optimized for expression in plant plastids (L1(pt) gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5'-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy.

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Transforming growth factor beta is a multi-functional growth and differentiation factor responsible for regulating many diverse biological processes in both vertebrate and invertebrate species. Among the most dramatic of TGFbeta's effects are those associated with specification of cell fates during development and inhibition of cell cycle progression. The core TGFbeta signaling pathway has now been described using a synergistic combination of genetic and biochemical approaches. Transmembrane receptors with intrinsic protein serine kinase activity bind ligand in the extracellular milieu and then phosphorylate intracellular proteins known as Smads. Phosphorylated Smads form heterooligomers and translocate into the nucleus where they can modulate transcriptional responses. More recent studies indicate that many other proteins serve as modulators of Smad activity, and utimately define specific cellular responses to TGFbeta. Here we describe both the simplistic core TGFbeta signaling pathway and the growing number of proteins that impinge on this pathway at the level of Smad function to either enhance or inhibit TGFbeta responses.

In this chapter we provide an overview of cytosine-to-uridine (C-to-U) RNA editing in the plastids of higher plants. Particular emphasis will be placed on the role plastid transformation played in understanding the editing process. We discuss how plastid transformation enabled identification of mRNA cis elements for editing and gave the first insight into the role of editing trans factors. The introduction will be followed by a protocol for plastid transformation, including vector design employed to identify editing cis elements. We also discuss how to test RNA editing in vivo by cDNA sequencing. At the end, we summarize the status of the field and outline future directions.

Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes.

The genes required for meiosis and sporulation in yeast are expressed at specific points in a highly regulated temporal pathway. Recent experiments using DNA microarrays to examine gene expression during meiosis and the identification of many regulatory factors have provided important advances in our understanding of how genes are regulated at the different stages of meiosis.

Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.

Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.

Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP-dependent promoters, the DNA target site for CAP is centred near position -42, overlapping and replacing the -35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is 'anti-inhibition,' overcoming an inhibitory effect of the RNAP alpha subunit C-terminal domain (alpha CTD). This component involves direct contact between amino acids 156-164 (activating region 1) of the upstream subunit of the CAP dimer and a target in alpha CTD. The second component is 'direct activation', facilitating isomerization of the RNAP-promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP alpha subunit N-terminal domain (alpha NTD).

Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex. Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.

The decapentaplegic gene complex (DPP-C) has been implicated in several events in pattern formation during Drosophila development. During embryogenesis, the DPP-C participates in the establishment of dorsal-ventral specification. Later, it is required for the correct morphogenesis of the imaginal disks, which will form much of the adult epidermis. We have undertaken a molecular analysis of the DPP-C to determine what role it plays in positional information. It appears to be a large genetic unit (greater than 40 kilobases (kb] consisting mostly of cis-regulatory information controlling the expression of a set of overlapping transcripts that differ at their 5' ends, but share the bulk of their transcribed sequences. Here, we describe the sequence analysis of two complementary DNAs comprising 4.0 kb of a 4.5-kb transcript. The C-terminus of the protein thereby deduced exhibits strong sequence homology (25-38% amino-acid identity) to the C-termini of a class of mammalian proteins that includes transforming growth factor-beta (TGF-beta), inhibin and Müllerian inhibiting substance (MIS). These proteins act on target cells to produce a variety of responses, such as stimulation or inhibition of cell division or differentiation. The homology suggests that the DPP-C protein contributes to correct morphogenesis as a secreted factor involved in the differential regulation of cell growth. This is the first report of a member of the TGF-beta gene family in a non-mammalian organism, and indicates that one or more members of this gene family existed before arthropod and vertebrate lineages diverged.

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, approximately 240 A channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of approximately 50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.

The Drosophila decapentaplegic (dpp) gene, a member of the transforming growth factor beta superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.