6 research outputs found
A Very Strong Enhancer Is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus
A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and −524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors
A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus
Using the simian virus 40 "enhancer trap"
approach, we have identified a transcription enhancer located
just upstream of the major immediate early gene of murine
cytomegalovirus. This enhancer has several striking properties.
(.) Together with the enhancer ofhuman cytomegalovirus,
it is the strongest transcription enhancer found to date. (ö) It
is an extremely long enhancer, spanning >700 base pairs. (ÜI)
It consists of a rather complex pattern of sequence repeats, the
longest of which is 181 base pairs. Also, several types of short
sequence motifs are scattered throughout the enhancer in
monomeric, heterodimeric, or homodimeric (palindromic)
form. These motifs have been identified to be components of
other enhancers and promoters, and they are presumably
binding sites for specific nuclear factors. Our analysis suggests
that enhancers are composed of a modular arrangement of
short conserved sequence motifs and that enhancer strength is
correlated with the redundancy of these motifs
The strong enhancer element in the immediate early region of the human cytomegalovirus genome
The human cytomegalovirus (HCMV), a
member of the herpesvirus group, was found to
possess a strong transcription enhancer in the
immediate early gene region. Co-transfection
of enhancerless SV40 DNA with randomly fragmented
HCMV DNA yielded two SV40-like recombinant
viruses , each containing HCMV DNA fragments that
were substituting for the missing SV40 enhancer.
The two inserts , 341 and 262 bp in length , are
overlapping segments of genuine viral DNA representing
part of the 5'flanking region of the
major immedistte early gene i n HCMV. Studies
with deletion mutants showed that different nonoverlapping
subsets of the HCMV enhancer region
can substitute for the 72 bp repeats of SV40.
Transient expression assays indicated that the
HCMV enhancer is significantly stronger than the
SV40 element, activating cis-linked heterologous
promoters in a wide spectrum of cultured cells.
It appears that the HCMV enhancer is positively
regulated by viral immediate early genes
L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway
Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be crucial for invasion and metastasis. Here, we investigate signal transduction leading to L-plastin Ser5 phosphorylation using 4 human breast cancer cell lines. Whole-genome microarray analysis comparing cell lines with different invasive capacities and corresponding variations in L-plastin Ser5 phosphorylation level revealed that genes of the ERK/MAPK pathway are differentially expressed. It is noteworthy that in vitro kinase assays showed that ERK/MAPK pathway downstream ribosomal protein S6 kinases α-1 (RSK1) and α-3 (RSK2) are able to directly phosphorylate L-plastin on Ser5. Small interfering RNA- or short hairpin RNA-mediated knockdown and activation/inhibition studies followed by immunoblot analysis and computational modeling confirmed that ribosomal S6 kinase (RSK) is an essential activator of L-plastin. Migration and invasion assays showed that RSK knockdown led to a decrease of up to 30% of migration and invasion of MDA-MB-435S cells. Although the presence of L-plastin was not necessary for migration/invasion of these cells, immunofluorescence assays illustrated RSK-dependent recruitment of Ser5-phosphorylated L-plastin to migratory structures. Altogether, we provide evidence that the ERK/MAPK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with RSK1 and RSK2 kinases able to directly phosphorylate L-plastin residue Ser5