Production of polyomavirus late mRNAs requires sequences near the 5' end of the leader but does not require leader-to-leader splicing.

Polyomavirus late mRNAs contain multiple copies of a 57-nucleotide leader sequence derived by splicing from multigenome-length late transcripts. Inefficient termination of transcription and inefficient polyadenylation allow accumulation of these giant transcripts. In this report, we show that a viable mutant virus, ins5, which contains an efficient rabbit beta-globin polyadenylation signal, produced late mRNAs whose vast majority contains only one leader. ins5 virus nevertheless produced as much late mRNA as did wild-type virus and grew as well as did wild-type virus in mouse cells. These results demonstrate that leader-to-leader splicing per se is not required for efficient production of late mRNAs or for efficient virus replication. However, we also found that RNAs lacking critical sequences near the 5' end of the leader did not accumulate as mRNAs and that most late transcripts made during the early part of the late phase, when few late mRNAs are produced, initiated downstream of the 5' end of the leader. These results indicate that a sequence element near the 5' end of the leader is required for proper processing, transport, or stability of late mRNAs and that the control of late mRNA production depends in part on the choice of transcription initiation sites at the late promoter.

[Anthropometric study on Down syndrome in Taiwan].

Anthropometric studies of Down syndrome in foreign children had been reported. To have similar information for Chinese children with Down syndrome, data based on 1624 measurements of height and 1208 measurements of weight were done on 496 children who took part in a collaborative study done via several hospitals and institutions in Taiwan. Height and weight were measured using standard methods. Additional data about these children were also solicited from hospital and school records to obtain semilongitudinal data which were then analyzed with SAS PC software. Descriptive statistics and percentiles were estimated using flexible mathematical functions. Results showed that ethnic Chinese children with Down syndrome were significantly shorter than normal children. Mean weight, however, was not significantly different from normal children. Centile charts for assessment of stature and weight are also presented.

Multiple domains in the polyomavirus B enhancer are required for productive infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.

Unique requirement for the PyF441 mutation for polyomavirus infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the enhancer of the polyomavirus host range mutant F441 permits productive infection of F9 embryonal carcinoma cells, which, when undifferentiated, are refractory to infection by wild-type polyomavirus. Synthetic oligonucleotides were used to construct viral genomes containing all four possible nucleotide pairs at nucleotide 5258. While all four of the viruses infected 3T6 cells efficiently, only F441, which has a guanosine in place of the wild-type adenosine in the early strand of DNA at position 5258, was able to infect F9 cells. Transfection assays with enhancer-dependent plasmid constructs expressing the chloramphenicol acetyltransferase gene under the control of the polyomavirus early promoter verified that only the F441 enhancer had any significant activity in F9 cells. DNase I footprinting showed that the F441 mutation creates a strong binding site for purified CCAAT box transcription factor, which is identical to nuclear factor 1. The three other mutations at nucleotide 5258 alter the affinity and the quality of factor binding at this site.

Duplication of functional polyadenylation signals in polyomavirus DNA does not alter efficiency of polyadenylation or transcription termination.

We constructed viable insertion mutants of polyomavirus that contain duplications of the nucleotide sequences surrounding the polyadenylation sites for both E- and L-strand RNAs. Our results showed that formation of poly(A)+ 3'termini of L-strand mRNAs requires sequence elements located between 12 and 87 nucleotides downstream of AAUAAA. No more than 19 nucleotides upstream and 44 nucleotides downstream of AAUAAA are required for polyadenylation of E-strand mRNAs. Our results and those of others suggest that there are three distinct sequence elements required for mRNA 3' end formation: AAUAAA and two downstream elements. An insertion mutant containing two adjacent functional polyadenylation signals produced E-strand and L-strand mRNAs with 3' ends at both sites. However, the overall level of polyadenylation of L-strand RNAs was not increased over the low (10 to 25%) levels seen with wild-type virus. Neither was the efficiency of termination of L-strand transcription increased in mutant virus-infected cells. We conclude that factors required for both polyadenylation and transcription termination are limiting in polyomavirus-infected mouse cells.

Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.