Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.

Activating transcription from single stranded DNA.

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.

DNA analysis and diagnostics on oligonucleotide microchips.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.

DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.

Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.

Relatedness threshold for the production of female sexuals in colonies of a polygynous ant, Myrmica tahoensis, as revealed by microsatellite DNA analysis.

The genetic relationships of colony members in the ant Myrmica tahoensis were determined on the basis of highly polymorphic microsatellite DNA loci. These analyses show that colonies fall into one of two classes. In roughly half of the sampled colonies, workers and female offspring appear to be full sisters. The remaining colonies contain offspring produced by two or more queens. Colonies that produce female sexuals are always composed of highly related females, while colonies that produce males often show low levels of nestmate relatedness. These results support theoretical predictions that workers should skew sex allocation in response to relatedness asymmetries found within colonies. The existence of a relatedness threshold below which female sexuals are not produced suggests a possible mechanism for worker perception of relatedness. Two results indicate that workers use genetic cues, not queen number, in making sex-allocation decisions. (i) The number of queens in a colony was not significantly correlated with either the level of relatedness asymmetry or the sex ratio. (ii) Sex-ratio shifts consistent with a genetically based mechanism of relatedness assessment were seen in an experiment involving transfers of larvae among unrelated nests. Thus workers appear to make sex-allocation decisions on the basis of larval cues and appear to be able to adjust sex ratios long after egg laying.

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences.

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.