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Darius Balciunas

Publications and source records attributed to Darius Balciunas.

9 recordsLinked to original sources
PubMed2006

The jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact with 19 proteins involved in transcription, sumoylation and DNA repair.

The jumonji domain is a highly conserved bipartite domain made up of two subdomains, jmjN and jmjC, which is found in many eukaryotic transcription factors. The jmjC domain was recently shown to possess the histone demethylase activity. Here we show that the jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact in a two-hybrid system with 19 yeast proteins that include the RecQ helicase Sgs1, the silencing factors Esc1 and Sir4, the URI-type prefoldin Bud27 and the PIAS type SUMO ligase Nfi1/Siz2. Extensive interaction cross dependencies further suggest that the proteins form a larger complex. Consistent with this, 16 of the proteins also interact with a Bud27 two-hybrid bait, and three of them co-precipitate with TAP-tagged Gis1. The Gis1 jumonji domain can repress transcription when recruited to a promoter as a lexA fusion. This effect is dependent on both the jmjN and jmjC subdomains, as were all 19 two-hybrid interactions, indicating that the two subdomains form a single functional unit. The human Sgs1 homolog WRN also interacts with the Gis1 jumonji domain. Finally, we note that several jumonji domain interactors are related to proteins that are found in mammalian PML nuclear bodies.

Exodeoxyribonucleases↗
PubMed2006

Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.

Animals↗
PubMed2006

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

Animals↗
PubMed2006

Functional and physical interactions within the middle domain of the yeast mediator.

Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserved in all eukaryotes. It is thought to play an important role in both transcriptional activation and repression. In the yeast Saccharomyces cerevisiae, Med21 is known to interact both with the Mediator subunit Med6 and the global co-repressor Tup1. We have made a temperature-sensitive med21-ts mutant, which we used in a high copy number suppressor screen. We found ten yeast genes that can suppress the med21-ts mutation in high copy number. The three strongest suppressors were MED7 and MED10 (NUT2), which encode other Mediator subunits, and ASH1, which encodes a repressor of the HO gene. 2-Hybrid experiments confirmed multiple interactions between Med21, Med10, Med7 and Med4, and also revealed a Med21 self-interaction. The interactions of Med21 with Med7 and Med10 were verified by co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, where both interactions were found to depend strongly on the amino acid residues 2-8 of Med21. These interactions, and the interactions of Med21 with Med6 and Tup1, suggest that Med21 may serve as a molecular switchboard that integrates different signals before they reach the core polymerase.

Amino Acid Sequence↗
PubMed2005

The structural and functional role of Med5 in the yeast Mediator tail module.

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5delta and med15delta deletions. The med5delta and med16delta deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.

Animals↗
PubMed2004

Enhancer trapping in zebrafish using the Sleeping Beauty transposon.

BACKGROUND: Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. RESULTS: We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. CONCLUSIONS: This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.

Animals↗
PubMed2003

Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon.

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

Animals↗
PubMed2002

Functional interactions within yeast mediator and evidence of differential subunit modifications.

It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms.

Base Sequence↗