Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7–11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

The Schizosaccharomyces pombe hst4+ Gene Is a SIR2 Homologue with Silencing and Centromeric Functions

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4+ gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Δ mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4+ appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Δ mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4+ is most similar to HST4. Furthermore, hst4+ is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4+ rescues the temperature-sensitivity and telomeric silencing defects of an hst3Δ hst4Δ double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.

Epistatic and independent functions of Caspase-3 and Bcl-XL in developmental programmed cell death

The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-XL directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-XL in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.

A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal

The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal–ventral axis in the Drosophila embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal–Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube–Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.

Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

New infinite families of exact sums of squares formulas, Jacobi elliptic functions, and Ramanujan’s tau function

In this paper, we give two infinite families of explicit exact formulas that generalize Jacobi’s (1829) 4 and 8 squares identities to 4n2 or 4n(n + 1) squares, respectively, without using cusp forms. Our 24 squares identity leads to a different formula for Ramanujan’s tau function τ(n), when n is odd. These results arise in the setting of Jacobi elliptic functions, Jacobi continued fractions, Hankel or Turánian determinants, Fourier series, Lambert series, inclusion/exclusion, Laplace expansion formula for determinants, and Schur functions. We have also obtained many additional infinite families of identities in this same setting that are analogous to the η-function identities in appendix I of Macdonald’s work [Macdonald, I. G. (1972) Invent. Math. 15, 91–143]. A special case of our methods yields a proof of the two conjectured [Kac, V. G. and Wakimoto, M. (1994) in Progress in Mathematics, eds. Brylinski, J.-L., Brylinski, R., Guillemin, V. & Kac, V. (Birkhäuser Boston, Boston, MA), Vol. 123, pp. 415–456] identities involving representing a positive integer by sums of 4n2 or 4n(n + 1) triangular numbers, respectively. Our 16 and 24 squares identities were originally obtained via multiple basic hypergeometric series, Gustafson’s Cℓ nonterminating 6φ5 summation theorem, and Andrews’ basic hypergeometric series proof of Jacobi’s 4 and 8 squares identities. We have (elsewhere) applied symmetry and Schur function techniques to this original approach to prove the existence of similar infinite families of sums of squares identities for n2 or n(n + 1) squares, respectively. Our sums of more than 8 squares identities are not the same as the formulas of Mathews (1895), Glaisher (1907), Ramanujan (1916), Mordell (1917, 1919), Hardy (1918, 1920), Kac and Wakimoto, and many others.

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown.

Identification of novel genes in Drosophila reveals the complex regulation of early gene activity in the mesoderm.

Two zygotic genes, twist and snail, are indispensable for the correct establishment of the mesoderm primordium in the early Drosophila embryo. They are also needed for morphogenesis and differentiation of the mesoderm. Both genes code for transcription factors with different, albeit complementary, functions. Therefore, to understand the early development of the mesoderm, it will be necessary to identify and study the genes regulated by twist and snail. We have searched for downstream genes using a subtractive cDNA library enriched in sequences expressed in the mesoderm. We have isolated sequences that correspond to 13 novel early mesoderm genes. These novel genes show a variety of expression patterns and also differ in their dependence on twist and snail functions. This indicates that the regulation of early gene activity in the mesoderm is more complex than previously thought.