Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Tissue-selective expression of dominant-negative proteins for the regulation of vascular smooth muscle cell proliferation.

The transcription factors c-myb and c-myc are essential for vascular smooth muscle cell (VSMC) replication and are rapidly induced following mitogenic stimulation of quiescent VSMCs in vitro and in vivo following balloon catheter injury. Consequently, interference with c-myb and c-myc function provides a possible avenue for the prevention of VSMC proliferation associated with intimal hyperplasia. We have carried out studies focused on the inhibition of VSMC proliferation using dominant-negative gene constructs incorporating the DNA-binding domains of the c-myb or c-myc genes fused to the repressor domain of the Drosophila engrailed gene. Transient transfection of rat, rabbit and human vascular SMCs results in a dramatic inhibition of proliferation for at least 72 h after transfection. Furthermore, this inhibition of cellular proliferation was found to be due, at least in part, to the induction of apoptosis. Coupling expression of the chimeric dominant-negative proteins to transcriptional regulatory elements of the human vascular smooth muscle alpha-actin gene allows specific targeting of vascular smooth muscle cells.

Efficient liposome-mediated gene transfer to rabbit carotid arteries in vivo.

Methods for gene delivery in vivo vary dramatically in their relative efficiencies. The most efficient to date involve the use of recombinant viruses, most notably adenoviruses, adeno-associated viruses (AAVs), or retroviruses (see Chapters 32 and 34 ). The creation of these recombinant constructs is laborious and requires specialized techniques, however. A technique that can efficiently deliver simple plasmids containing the gene of interest in vivo allows the screening of gene constructs to determine their effects prior to embarking on the creation of recombinant viruses.

A function for CD2 on murine B cells?

CD2 is expressed on murine B cells, probably as a result of a chromosomal translocation event during speciation. There are no activating antibodies to mouse CD2, but when activated non-specifically, only T cells up-regulate their expression of CD2. We investigated the expression and function of CD2 on B cells using mice transgenic for human CD2 under the control of a modified version of the autologous promoter/enhancer that included the immunoglobulin enhancer. This construction directs expression of human CD2 to the B cell compartment as well as to the T cell compartment. In this paper, we confirm that activating pairs of anti-CD2 monoclonal antibodies are mitogenic for mouse T cells transgenic for human CD2. In contrast, mouse B cells that express similar amounts of human CD2 are not stimulated to proliferate by equivalent doses of these antibodies. We were also unable to show any functional consequence for these B cells as a result of CD2 ligation.

High efficiency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex.

Efficient transfection conditions for a number of human, rat and rabbit primary cells and established lines of vascular origin have been determined using a complex of a commercially available cationic lipid transfection agent (Tfx-50) and luciferase reporter plasmid constructs. The optimised conditions have also been successfully applied to rabbit carotid arteries in vivo and a series of human arteries in vitro. The most critical factors influencing the efficiency of gene transfection with this protocol are: DNA concentration; ratio of lipid reagent to DNA; transfection time and the presence or absence of serum. Immunohistochemical analysis shows that a high percentage of cells (approximately 30-80% dependent on lineage) were transfected under optimal conditions with minimal toxicity effects. Similar analyses performed on undamaged rabbit carotid vessels transfected in vivo and human arteries transfected in vitro show high-efficiency transfer and strong expression of the luciferase vector as demonstrated by reporter gene expression. The optimisation of gene transfer into vascular cells with this cationic lipid complex will be valuable for molecular studies of genes implicated in cardiovascular diseases and as a possible method of gene delivery with therapeutic intent.