Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF– and the derivative mutant–microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.

Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis

The transcription of CAB genes, encoding the chlorophyll a/b-binding proteins, is rapidly induced in dark-grown Arabidopsis seedlings following a light pulse. The transient induction is followed by several cycles of a circadian rhythm. Seedlings transferred to continuous light are known to exhibit a robust circadian rhythm of CAB expression. The precise waveform of CAB expression in light–dark cycles, however, reflects a regulatory network that integrates information from photoreceptors, from the circadian clock and possibly from a developmental program. We have used the luciferase reporter system to investigate CAB expression with high time resolution. We demonstrate that CAB expression in light-grown plants exhibits a transient induction following light onset, similar to the response in dark-grown seedlings. The circadian rhythm modulates the magnitude and the kinetics of the response to light, such that the CAB promoter is not light responsive during the subjective night. A signaling pathway from the circadian oscillator must therefore antagonize the phototransduction pathways controlling the CAB promoter. We have further demonstrated that the phase of maximal CAB expression is delayed in light–dark cycles with long photoperiods, due to the entrainment of the circadian oscillator. Under short photoperiods, this pattern of entrainment ensures that dawn coincides with a phase of high light responsiveness, whereas under long photoperiods, the light response at dawn is reduced.

Positive and negative regulation of endogenous genes by designed transcription factors

Gene regulation by imposed localization was studied by using designed zinc finger proteins that bind 18-bp DNA sequences in the 5′ untranslated regions of the protooncogenes erbB-2 and erbB-3. Transcription factors were generated by fusion of the DNA-binding proteins to repression or activation domains. When introduced into cells these transcription factors acted as dominant repressors or activators of, respectively, endogenous erbB-2 or erbB-3 gene expression. Significantly, imposed regulation of the two genes was highly specific, despite the fact that the transcription factor binding sites targeted in erbB-2 and erbB-3 share 15 of 18 nucleotides. Regulation of erbB-2 gene expression was observed in cells derived from several species that conserve the DNA target sequence. Repression of erbB-2 in SKBR3 breast cancer cells inhibited cell-cycle progression by inducing a G1 accumulation, suggesting the potential of designed transcription factors for cancer gene therapy. These results demonstrate the willful up- and down-regulation of endogenous genes, and provide an additional means to alter biological systems.

A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans

The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.

Mechanism of Ca2+-dependent nuclear accumulation of calmodulin

The intracellular Ca2+ receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca2+-dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca2+-dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca2+-dependence and affinity. At high [Ca2+], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.

WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: The proline glycine and methionine-rich motif

Pre-mRNA splicing requires the bridging of the 5′ and 3′ ends of the intron. In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP. Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP’s), each of which contains one or more of a special class of tyrosine-rich WW domains. Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40. We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB′, and the branchpoint binding protein SF1/mBBP. Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex.

HNS, a nuclear-cytoplasmic shuttling sequence in HuR

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3′ untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.

Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix–loop–helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.

A nonnatural transcriptional coactivator

In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.

A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of β-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-Å resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type d-alanyl-d-alanine carboxypeptidase/transpeptidase (EC 3.4.16.4) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

p53 associates with and targets ΔNp63 into a protein degradation pathway

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation.

Protein–RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.

Fluorescent RNA cytochemistry: tracking gene transcripts in living cells

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of ‘fluorescent RNA cytochemistry’ is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the investigation of RNA birth, RNA–protein assembly and ribonucleoprotein particle transport in systems such as oocytes, embryos, neurons and other somatic cells, and may even permit the observation of viral replication and transcription pathways as they proceed in living cells, ushering in a new era of nucleic acids research in vivo.

Identification and dissection of an enhancer controlling epithelial gene expression in skin

Keratins 14 and 5 are the structural hallmarks of the basal keratinocytes of the epidermis and outer root sheath (ORS) of the hair follicle. Their genes are controlled in a tissue-specific manner and thus serve as useful tools to elucidate the regulatory mechanisms involved in keratinocyte-specific transcription. Previously we identified several keratinocyte-specific DNase I hypersensitive sites (HSs) in the 5′ regulatory sequences of the K14 gene and showed that a 700-bp regulatory domain encompassing HSs II and III can confer epidermal and ORS-specific gene expression in transgenic mice in vivo. Although HS II harbored much of the transactivation activity in vitro, it was not sufficient to restrict expression to keratinocytes in vivo. We now explore the HS III regulatory element. Surprisingly, this element on its own confers gene expression to the keratinocytes of the inner root sheath (IRS) of the hair follicle, whereas a 275-bp DNA fragment containing both HSs II and III shifts the expression from the IRS to the basal keratinocytes and ORS in vivo. Electrophoretic mobility-shift assays and mutational studies of HSs III reveal a role for CACCC-box binding proteins, Sp1 family members, and other factors adding to the list of previously described factors that are involved in keratinocyte-specific gene expression. These studies highlight a cooperative interaction of the two HSs domains and strengthen the importance of combinatorial play of transcription factors that govern keratinocyte-specific gene regulation.