Mapping 5'' termini of JC virus early RNAs.

Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element.     

Human polyomavirus JC virus genome.

The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.