Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Lipopolyamine-Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells

A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.     

Unraveling the early steps of prokaryotic replication

In prokaryotes, many of the physical mechanisms governing the process of initiating DNA replication are now emerging. For example, certain organizational features of origins, such as the use of repetitive sequence elements for initiator-binding sites, are found throughout bacteria and many archaea. Common themes in the regulation of initiation, including origin sequestration by trans-acting factors, titration of initiator levels by cis- and trans-acting factors, and control of initiator function by ATP, also exist. Recent studies have shown that prokaryotic initiator structures are both modular and conserved, and have begun to reveal how these proteins specifically recognize target DNA sequences. These properties probably control initiator self-assembly and DNA remodeling to properly fire replication origins.     

Chimeric genes and transgenic plants are used to study the regulation of genes involved in symbiotic plant-microbe interactions (nodulin genes)

Nodulin genes are plant genes specifically activated during the formation of nitrogen-fixing nodules on leguminous plants. These genes are interesting to study since they are not only induced in a specific developmental fashion by signals coming directly or indirectly from the rhizobial symbiont, but are also expressed in a tissue-specific manner. By examining the expression of chimeric nodulin-reporter genes in transgenic legume plants it has been shown that nodule specific expression is mediated by DNA sequences present in the 5 upstream region of several nodulin genes. Here we summarize the available data on these cis-acting elements and the trans-acting factors interacting with them. We also review experiments designed to identify rhizobial "signals" which may play a role in nodule specific gene expression.     

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.     

Regulation of imprinted DNA methylation

DNA methylation is an essential enzymatic modification in mammals. This common epigenetic mark occurs predominantly at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3'. The majority of methylated CpGs are located within repetitive elements including centromeric repeats, satellite sequences and gene repeats encoding ribosomal RNAs. CpG islands, frequently located at the 5' end of genes, are typically unmethylated. DNA methylation also occurs at imprinted genes which exhibit parent-of-origin-specific patterns of methylation and expression. Imprinted methylation at differentially methylated domains (DMDs) is one of the regulatory mechanisms controlling the allele-specific expression of imprinted genes. Proper control of DNA methylation is needed for normal development and loss of methylation control can contribute to initiation and progression of tumorigenesis (reviewed in Plass and Soloway, 2002). Because patterns of imprinted DNA methylation are highly reproducible, imprinted loci make useful models for studying regulation of DNA methylation and may provide insights into how this regulation goes awry in cancer. Here, we review what is currently known about the mechanisms regulating imprinted DNA methylation. We will focus on cis-acting DNA sequences, trans-acting protein factors and the possible involvement of RNAs in control of imprinted DNA methylation.     

A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos

AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15.     

Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage λ

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.     

G1 events and the regulation of genes for S-phase enzymes.

The genes for S-phase enzymes are expressed at low levels in quiescent mammalian cells but at high levels during DNA replication. Regulation occurs at multiple levels by mechanisms that are different for each gene. Current research is focused on identifying the control elements and trans-acting factors for each gene and establishing relationships between these regulatory mechanisms and the G1 signal transduction pathway.     

Negative trans-acting factors extinguish Ia expression in B cell-L 929 somatic cell hybrids

Numerous studies have implicated trans-acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II genes while others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans-acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role trans-acting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk- cell line and normal B cells to determine if the existence of positive trans-acting factors from the B cell would lead to the expression of Ltk- class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk- class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggest that Ltk- cells possess negative trans-acting factors that appear to predominate over the positive trans-acting factors possessed by B cells. We have further extended these studies to test the MHC-inducing activity of IFN-gamma and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN-gamma with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II genes were induced by IL-4. Taken together, these results indicate that Ltk- cells possess negative trans-acting factors that not only maintain the Ia- phenotype of these cells, but also block the action of positive trans-acting factors from B cells.