Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

Unwinding of the third strand of a DNA triple helix, a novel activity of the SV40 large T-antigen helicase.

We present experiments indicating that the SV40 large T-antigen (T-ag) helicase is capable of unwinding the third strand of DNA triple helices. Intermolecular d(TC)(20)d(GA)(20)d(TC)(20) triplexes were generated by annealing, at pH 5.5, a linearized double-stranded plasmid containing a d(TC)(27).d(GA)27 tract with a (32)P-labeled oligonucleotide consisting of a d(TC)(20) tract flanked by a sequence of 15 nt at the 3'-end. The triplexes remained stable at pH 7.2, as determined by agarose gel electrophoresis and dimethyl sulfate footprinting. Incubation with the T-ag helicase caused unwinding of the d(TC)(20) tract and consequent release of the oligonucleotide, while the plasmid molecules remained double-stranded. ATP was required for this reaction and could not be replaced by the non-hydrolyzable ATP analog AMP-PNP. T-ag did not unwind similar triplexes formed with oligonucleotides containing a d(TC)(20) tract and a 5' flanking sequence or no flanking sequence. These data indicate that unwinding of DNA triplexes by the T-ag helicase must be preceded by binding of the helicase to a single-stranded 3' flanking sequence, then the enzyme migrates in a 3'--> 5' direction, using energy provided by ATP hydrolysis, and causes release of the third strand. Unwinding of DNA triplexes by helicases may be required for processes such as DNA replication, transcription, recombination and repair.

Forensic demography and civil rights.

In the summer of 1965, the legislature of the state of Mississippi passed a law which required children whose parents were living in another state to pay up to $350 tuition in order to attend public school. On the belief that this law discriminated against Negroes and was therefore unconstitutional, the Department of Justice began litigation procedures to repeal it. But evidence was needed to prove that the law was in fact discriminatory. The lawyers contacted demographers at the Bureau of the Census to provide data to determine if the majority of persons affected were Negro and too poor to pay the tuition. They sought information which could tell them, ideally, how many parents of children in Mississippi had left the state and what the economic position and racial characteristics of the children were.An attempt was made to get this information from already published sources, such as census reports and other research findings. These could give a hurried impression of the situation but could not deal directly with the issue of the whereabouts of parents not living with their children, since such a question had not been asked in the 1960 Census. An additional bit of information was obtained from the NAAGP Legal Defense and Education Fund in Jackson, Mississippi, from forms showing characteristics of persons actually affected by the law. But this limited sample did not provide evidence solid enough upon which to build a case.It was decided to rely on a retabulation of 1960 Census records to describe accurately the base population from which children with parents out of state would be drawn-namely, the children not living with either parent. This showed that of all school-age children living without either parent, 82 percent are Negro. The average Negro household, which contains at least one "parentless" child, houses 6.8 persons and has an annual income of $1,429. This allows less than $250 annually per person. Tuition for just one child eats up more than one person's yearly allotment. These data showed that the law, in fact, did discriminate racially, since most of the persons affected were Negro and too poor to pay tuition.This was one instance in which demographers were able to use their special skills in implementing public policy-notably, the law.