Rat antizyme gene expression requires programmed, ribosomal frameshifting. A novel autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of polyamines. Antizyme binds to, and destabilizes, ornithine decarboxylase, a key enzyme in polyamine synthesis. Rapid degradation ensues, thus completing a regulatory circuit. In vitro experiments with a fusion construct using reticulocyte lysates demonstrate polyamine-dependent expression with a frameshift efficiency of 19% at the optimal concentration of spermidine. The frameshift is +1 and occurs at the codon just preceding the terminator of the initiating frame. Both the termination codon of the initiating frame and a pseudoknot downstream in the mRNA have a stimulatory effect. The shift site sequence, UCC-UGA-U, is not similar to other known frameshift sites. The mechanism does not seem to involve re-pairing of peptidyl-tRNA in the new frame but rather reading or occlusion of a fourth base.

Selenoprotein P (Se-P) contains most of the selenium in plasma. Its function is not known. Mice with the Se-P gene deleted (Sepp(-/-)) were generated. Two phenotypes were observed: 1) Sepp(-/-) mice lost weight and developed poor motor coordination when fed diets with selenium below 0.1 mg/kg, and 2) male Sepp(-/-) mice had sharply reduced fertility. Weanling male Sepp(+/+), Sepp(+/-), and Sepp(-/-) mice were fed diets for 8 weeks containing <0.02-2 mg selenium/kg. Sepp(+/+) and Sepp(+/-) mice had similar selenium concentrations in all tissues except plasma where a gene-dose effect on Se-P was observed. Liver selenium was unaffected by Se-P deletion except that it increased when dietary selenium was below 0.1 mg/kg. Selenium in other tissues exhibited a continuum of responses to Se-P deletion. Testis selenium was depressed to 19% in mice fed an 0.1 mg selenium/kg diet and did not rise to Sepp(+/+) levels even with a dietary selenium of 2 mg/kg. Brain selenium was depressed to 43%, but feeding 2 mg selenium/kg diet raised it to Sepp(+/+) levels. Kidney was depressed to 76% and reached Sepp(+/+) levels on an 0.25 mg selenium/kg diet. Heart selenium was not affected. These results suggest that the Sepp(-/-) phenotypes were caused by low selenium in testis and brain. They strongly suggest that Se-P from liver provides selenium to several tissues, especially testis and brain. Further, they indicate that transport forms of selenium other than Se-P exist because selenium levels of all tissues except testis responded to increases of dietary selenium in Sepp(-/-) mice.

the members of the DOE and NIH planning groups R E V I E W The Human Genome Project has successfully completed all the major goals in its current 5-year plan, covering the period 1993-98. A new plan, for 1998 -2003, is presented, in which human DNA sequencing will be the major emphasis. An ambitious schedule has been set to complete the full sequence by the end of 2003, 2 years ahead of previous projections. In the course of completing the sequence, a "working draft" of the human sequence will be produced by the end of 2001. The plan also includes goals for sequencing technology development; for studying human genome sequence variation; for developing technology for functional genomics; for completing the sequence of Caenorhabditis elegans and Drosophila melanogaster and starting the mouse genome; for studying the ethical, legal, and social implications of genome research; for bioinformatics and computational studies; and for training of genome scientists.The Human Genome Project (HGP) is fulfilling its promise as the single most important project in biology and the biomedical sciences-one that will permanently change biology and medicine. With the recent completion of the genome sequences of several microorganisms, including Escherichia coli and Saccharomyces cerevisiae, and the imminent completion of the sequence of the metazoan Caenorhabditis elegans, the door has opened wide on the era of whole genome science. The ability to analyze entire genomes is accelerating gene discovery and revolutionizing the breadth and depth of biological questions that can be addressed in model organisms. These exciting successes confirm the view that acquisition of a comprehensive, high-quality human genome sequence will have unprecedented impact and long-lasting value for basic biology, biomedical research, biotechnology, and health care. The transition to sequence-based biology will spur continued progress in understanding gene-environment interactions and in development of highly accurate DNA-based medical diagnostics and therapeutics.Human DNA sequencing, the flagship endeavor of the HGP, is entering its decisive phase. It will be the project's central focus during the next 5 years. While partial subsets of the DNA sequence, such as expressed sequence tags (ESTs), have proven enormously valuable, experience with simpler organisms confirms that there can be no substitute for the complete genome sequence. In order to move vigorously toward this goal, the crucial task ahead is building sustainable capacity for producing publicly available DNA sequence. The full and incisive use of the human sequence, including comparisons to other vertebrate genomes, will require further increases in sustainable capacity at high accuracy and lower costs. Thus, a high-priority commitment to develop and deploy new and improved sequencing technologies must also be made.Availability of the human genome sequence presents unique scientific opportunities, chief among them the study of natural genetic variation in humans. Genetic or DNA sequence variat...

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Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.

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A minority of genes in probably all organisms rely on "recoding" for translation of their mRNAs. In these cases, the rules for decoding are temporarily altered through the action of specific signals built into the mRNA sequences. Three classes are described. 1. Frameshifting at a particular site allows expression of a protein from an mRNA with overlapping open reading frames, often giving two protein products from one mRNA. 2. The meanings of code words are altered: specific stop codons can be redirected to encode selenocysteine, tryptophan, or glutamine. 3. Ribosomes can translate over coding gaps in mRNA. These novel mechanisms expand the repertoire of the genetic code and are at the heart of several regulatory schemes.

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