Adenoviral-mediated gene transfer to bladder in vivo.

This study was designed to examine the potential for gene therapy in bladder in vivo using adenoviral vectors. Gene transfer to rat bladders was accomplished via direct intravesical instillation using a replication-defective adenoviral vector containing a marker gene encoding for Escherichia coli beta-galactosidase (beta-gal). Successful gene transfer was confirmed by analyzing bladder samples for DNA and RNA using polymerase chain reaction (PCR) with primers specific for beta-gal and adeno sequences, detecting beta-gal in full-thickness bladder wall using specific histochemical staining (X-gal) and documenting recombinant protein production. Bladder architecture was preserved, without evidence of distant spread of virus as assessed by PCR. Gene expression was evident for at least 7 days. In summary, bladder cells can be genetically altered using replication-deficient adenoviral vectors via simple intravesical instillation of vector. Introduction of exogenous genetic material is a potentially powerful therapeutic modality for immunomodulation of bladder neoplasms.

The 5'-limit of transposition and upstream barren region of a trypanosome VSG gene: tandem 76 base-pair repeats flanking (TAA)90.

We have cloned and sequenced a portion of the region upstream of an expressed VSG gene of Trypanosoma brucei. This "expression-linked copy" arose through the duplication and transposition of a silent, "basic copy" of the gene to an expression site. Comparison of the sequences surrounding the 5'-end of the transposed segment in the two loci indicates the 5'-limit of transposition lies within the first (3'-most) of three repeated segments found at this position in the basic copy locus. These highly conserved repeat segments which average 76 base-pairs in length are also found tandemly repeated upstream of the transposed segment in the expression site. In this latter site, however, they are more numerous (at least 17 repeats) and they are interrupted, within the middle of one repeat, by a 270 base-pair region consisting of (TAA)90. The possible roles of these unusual sequences in transposition and in a model proposing discontinuous transcription of VSG genes are discussed.

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7.

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.

The nucleotide sequence of the gene encoding protein IVa2 in human adenovirus type 7.

The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined. When compared to Ad5, a group C adenovirus, these sequences have been found to be 80% homologous. Most changes are transitions or transversions. This high degree of nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded from these genes; most nucleotide changes occur at the third position in the codon. The predicted polypeptide contains 448 amino acids and has a calculated Mr-value of 50700. The positions of the 5' end of the mRNA and of the donor and acceptor splice sites of Ad7 and Ad12 can be inferred by analogy to those of Ad5. A long open reading frame starting upstream from the IVa2 gene overlaps the N-terminal portion of the polypeptide but is encoded in a different reading frame. Within this overlapping region, the long open reading frame is more conserved in amino acid sequence than is the presumed IVa2 polypeptide, suggesting that evolutionary pressure was exerted on the longer protein, a product of viral early region 2B. The high degree of conservation of this E2B region within the overlapping segment suggests that its activities must be more important for adenovirus infection than are the functions encoded in the amino-terminus of the IVa2 gene.

The nucleotide sequence and protein-coding capability of the transposable element IS5.

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.