49,212 research outputs found
Transferrin-polycation conjugates as carriers for DNA uptake into cells.
We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection.
Cell-type specificity of regulatory elements identified by linker scanning mutagenesis in the promoter of the chicken lysozyme gene
The chicken lysozyme gene is constitutively expressed in macrophages, in oviduct cells its expression is controlled by steroid hormones, and in fibroblasts the gene is not expressed. A fusion gene consisting of promoter sequences of the lysozyme gene from –208 to +15 in front of the chloramphenicol acetyltransferase (CAT) coding region was more than 50 times less active in non-expressing cells as compared to expressing cells. In order to identify the element(s) responsible for this cell-type specificity 31 different linker scanning mutations were generated within this promoter fragment and analyzed by transient transfections in the three types of chicken cells mentioned above. Three mutation sensitive regions located around position –25, –100 and between –158 and –208 were detected in each cell type, however, several LS mutations displayed clear cell-type specific differences in their phenotypic effects. Interestingly, a few LS mutations led to an increase in promoter activity in fibroblasts suggesting that the corresponding wildtype sequences represent binding sites for negatively acting transcription factors
Gallus GBrowse: a unified genomic database for the chicken
Gallus GBrowse (http://birdbase.net/cgi-bin/gbrowse/gallus/) provides online access to genomic and other information about the chicken, Gallus gallus. The information provided by this resource includes predicted genes and Gene Ontology (GO) terms, links to Gallus In Situ Hybridization Analysis (GEISHA), Unigene and Reactome, the genomic positions of chicken genetic markers, SNPs and microarray probes, and mappings from turkey, condor and zebra finch DNA and EST sequences to the chicken genome. We also provide a BLAT server (http://birdbase.net/cgi-bin/webBlat) for matching user-provided sequences to the chicken genome. These tools make the Gallus GBrowse server a valuable resource for researchers seeking genomic information regarding the chicken and other avian species
The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken
The replacement histone H2A.Z is variously reported
as being linked to gene expression and preventing the
spread of heterochromatin in yeast, or concentrated
at heterochromatin in mammals. To resolve this
apparent dichotomy, affinity-purified antibodies
against the N-terminal region of H2A.Z, in both a triacetylatedandnon-
acetylatedstate, areusedin native
chromatin immmuno-precipitation experiments with
mononucleosomes from three chicken cell types. The
hyperacetylated species concentrates at the 50 end of
active genes, both tissue specific and housekeeping
but is absent from inactive genes, while the
unacetylated form is absent from both active and
inactive genes. A concentration of H2A.Z is also
found at insulators under circumstances implying a
link to barrier activity but not to enhancer blocking.
Although acetylated H2A.Z is widespread throughout
the interphase genome, at mitosis its acetylation is
erased, the unmodified form remaining. Thus,
although H2A.Z may operate as an epigenetic marker
for active genes, its N-terminal acetylation does not
The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken
The replacement histone H2A.Z is variously reported
as being linked to gene expression and preventing the
spread of heterochromatin in yeast, or concentrated
at heterochromatin in mammals. To resolve this
apparent dichotomy, affinity-purified antibodies
against the N-terminal region of H2A.Z, in both a triacetylatedandnon-
acetylatedstate, areusedin native
chromatin immmuno-precipitation experiments with
mononucleosomes from three chicken cell types. The
hyperacetylated species concentrates at the 50 end of
active genes, both tissue specific and housekeeping
but is absent from inactive genes, while the
unacetylated form is absent from both active and
inactive genes. A concentration of H2A.Z is also
found at insulators under circumstances implying a
link to barrier activity but not to enhancer blocking.
Although acetylated H2A.Z is widespread throughout
the interphase genome, at mitosis its acetylation is
erased, the unmodified form remaining. Thus,
although H2A.Z may operate as an epigenetic marker
for active genes, its N-terminal acetylation does not
A new method for constructing linker scanning mutants
A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from –208 to +1
TranspoGene and microTranspoGene: transposed elements influence on the transcriptome of seven vertebrates and invertebrates
Transposed elements (TEs) are mobile genetic sequences. During the evolution
of eukaryotes TEs were inserted into active protein-coding genes, affecting
gene structure, expression and splicing patterns, and protein sequences.
Genomic insertions of TEs also led to creation and expression of new functional
non-coding RNAs such as micro- RNAs. We have constructed the TranspoGene
database, which covers TEs located inside proteincoding genes of seven species:
human, mouse, chicken, zebrafish, fruit fly, nematode and sea squirt. TEs were
classified according to location within the gene: proximal promoter TEs,
exonized TEs (insertion within an intron that led to exon creation), exonic TEs
(insertion into an existing exon) or intronic TEs. TranspoGene contains
information regarding specific type and family of the TEs, genomic and mRNA
location, sequence, supporting transcript accession and alignment to the TE
consensus sequence. The database also contains host gene specific data: gene
name, genomic location, Swiss-Prot and RefSeq accessions, diseases associated
with the gene and splicing pattern. In addition, we created microTranspoGene: a
database of human, mouse, zebrafish and nematode TEderived microRNAs. The
TranspoGene and micro- TranspoGene databases can be used by researchers
interested in the effect of TE insertion on the eukaryotic transcriptome
AIDA: ab initio domain assembly server.
AIDA: ab initio domain assembly server, available at http://ffas.burnham.org/AIDA/ is a tool that can identify domains in multi-domain proteins and then predict their 3D structures and relative spatial arrangements. The server is free and open to all users, and there is an option for a user to provide an e-mail to get the link to result page. Domains are evolutionary conserved and often functionally independent units in proteins. Most proteins, especially eukaryotic ones, consist of multiple domains while at the same time, most experimentally determined protein structures contain only one or two domains. As a result, often structures of individual domains in multi-domain proteins can be accurately predicted, but the mutual arrangement of different domains remains unknown. To address this issue we have developed AIDA program, which combines steps of identifying individual domains, predicting (separately) their structures and assembling them into multiple domain complexes using an ab initio folding potential to describe domain-domain interactions. AIDA server not only supports the assembly of a large number of continuous domains, but also allows the assembly of domains inserted into other domains. Users can also provide distance restraints to guide the AIDA energy minimization
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