Joseph Shlomai

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

ABSTRACT Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

Trypanosomes Have Six Mitochondrial DNA Helicases, with One Controlling Kinetoplast Maxicircle Replication

Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma bruceis genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.

Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradients.

Herpes simplex virus (HSV) DNA molecules were isolated from infected BSC 1 cells and centrifuged in CsCl-ethidium bromide density gradients. Both newly labelled and mature virus DNA molecules were found to have a linear conformation. The morphology of virus DNA molecules at different stages of the virus growth cycle in BSC 1 cells, was studied by electron microscopy after separation of virus DNA from cellular DNA by centrifugation in CsCl gradients. In each sample, about 200 virus DNA molecules were photographed and the different morphological forms were studied. Four classes of virus DNA molecules were observed: (a) mature linear DNA molecules, 52-4 +/- 3-3 micronm in length, (b) DNA intermediates, (c) virus DNA molecules having one or more single-stranded filaments attached to them and (d) molecules with collapsed regions or with branches. A few circular molecules as well as linear DNA molecules longer than unit length were also observed. The virus DNA molecules resembling replicative intermediates gradually increased in number and reached a maximal amount of about 5% of the virus DNA population at 12 h after infection. The other forms of virus DNA were found to persist after the number of replicating DNA molecules decreased.

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids PURIFICATION AND CHARACTERIZATION

Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5′-GGGGTTGGTGTA-3′). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.

Replicative intermediates of herpes simplex virus DNA

Abstract The DNA synthesized in herpes simplex virus (HSV)-infected nuclei in vivo was analyzed by chromatography on benzoylated naphthoylated DEAE-cellulose columns. Viral-DNA molecules that elute with caffeine contain single-stranded DNA sequences sensitive to a micrococcal endonuclease that cleaves single-stranded DNA only. These viral-DNA molecules behave kinetically as precursors to mature virion DNA, and their transition to the mature double-stranded form occurs within a period of 10 to 20 min. Electron microscopy revealed viral-DNA molecules at different stages of replication. The mechanism of HSV-DNA replication is discussed.

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.

Universal Minicircle Sequence-binding Protein, a Sequence-specific DNA-binding Protein That Recognizes the Two Replication Origins of the Kinetoplast DNA Minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5′-ACGCCC-3′) that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatidCrithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339–21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification fromC. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

Assigning functions to genes: identification of S-phase expressed genes in Leishmania major based on post-transcriptional control elements

Assigning functions to genes is one of the major challenges of the post-genomic era. Usually, functions are assigned based on similarity of the coding sequences to sequences of known genes, or by identification of transcriptional cis-regulatory elements that are known to be associated with specific pathways or conditions. In trypanosomatids, where regulation of gene expression takes place mainly at the post-transcriptional level, new approaches for function assignment are needed. Here we demonstrate the identification of novel S-phase expressed genes in Leishmania major, based on a post-transcriptional control element that was recognized in Crithidia fasciculata as involved in the cell cycle-dependent expression of several nuclear and mitochondrial S-phase expressed genes. Hypothesizing that a similar regulatory mechanism is manifested in L.major, we have applied a computational search for similar control elements in the genome of L.major. Our computational scan yielded 132 genes, of which 33% are homologues of known DNA metabolism genes and 63% lack any annotation. Experimental testing of seven of these genes revealed that their mRNAs cycle throughout the cell cycle, reaching a maximum level during S-phase or just prior to it. It is suggested that screening for post-transcriptional control elements associated with a specific function provides an efficient method for assigning functions to trypanosomatid genes.