Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. transcription from promoters in the intergenic region (IR) (4) and rolling-circle replication (5-8). Apart from one single viral protein, geminivirus DNA multiplication entirely relies on the DNA-replication apparatus of the host plant, representing a simple system to study DNA replication in the plant nucleus. The origin of viral-strand DNA replication is located in the IR and has been mapped to the conserved nonanucleotide sequence TAATATTAC, flanked by inverted repeats potentially forming a stem-loop (9, 10).Only one viral protein is indispensable for replication (11). This replication protein (AL1, Cl, or ORFIII/IV protein) is encoded by either one or two open reading frames. In the latter case, the mRNA spanning the overlapping reading frames is spliced to give rise to a single polypeptide chain (12,13 (27) was cloned in pGEX-3X (29). Alternatively to the factor Xa cleavage site of pGEXC1, the amino acid sequence-PGSGSGDDDK-specifying an enterokinase cleavage site (30) was engineered between the glutathione S-transferase (GST) and the Rep domains. The following oligonucleotides, 5'-GT GGGATCCCGGGCTCCGGCTCCGGCGACGACG-ACGACAAAATGCCAAGATCAGGTCGTTT-3' and 5'-C-ACCCTCAATCACTATACTCACCGG-3', were used for PCR (31) on pGEXC1 DNA, yielding the expression vector pGEXEC1.Expression of Rep Protein in E. coli and Purification. A 1:10 dilution of E. coli DH5 a (pGEXC1 or pGEXEC1) in NZCY medium (29) containing 100 ,tg of ampicillin per ml was grown for 1 hr at 28°C, and protein expression was induced by 0. (Fig. 1A) were done with the GST-Rep fusion of pGEXC1; all other experiments were done with the GSTRep fusion protein expressed by pGEXEC1. Approximately 300 ng of GST-Rep protein were incubated with substrate DNA in a total volume of 20 ,ul for 30 min at 37°C in cleavage buffer (25 mM Tris-HCl, pH 7.5/75 mM NaCl/5 mM MgCl2/ 2.5 mM dithiothreitol/0.5 mM EDTA), containing 50 fmol of each oligonucleotide or 100 fmol of double-stranded BspEIBamHI IR fragment. The reaction was stopped by 2 ,ul of 0.5 M EDTA, lyophilized, resuspended in loading buffer [98% (vol/vol) formamide] and analyzed on 12% sequencing gels, which were dried and autoradiographed. The cleavage products were excised from the gels and quantified by liquid scintillation counting.Abbreviations: GST, glutathione S-transferase; IR, intergenic region; Rep protein, replication initiator protein; TYLCV, tomato yellow leaf curl virus. tTo whom reprint requests should be addressed.

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.

No abstract

Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5‐kb mRNA with an abundance of 1–3 x 107 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5‐kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5′‐end of the transcript was determined by Northern hybridization and S1 mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUG‐free leader 600‐700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3′‐sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the first AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.

Both exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene individually stimulate expression of reporter genes in transient gene expression experiments if present within the transcription unit. The Sh1 exon 1 mediates a 10-fold increase in activity when inserted at the 5' end of the bacterial chloramphenicol transacetylase (CAT) marker gene in both monocot and dicot protoplasts. The Sh1 intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits CAT expression in tobacco protoplasts. In combination, the stimulatory effects of Sh1 exon 1 and intron 1 are multiplicative in monocot protoplasts resulting in a final enhancement of up to 1000-fold compared to the unmodified CAT or luciferase marker genes.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'rolling circle replication initiator protein', the M(r) 41 kDa viral Rep protein, encoded by the C1 or AC1 genes. Using wheat dwarf virus (WDV) and tomato yellow leaf curl virus (TYLCV) as examples, we show that not only the full-size Rep proteins, but also a putative 30 kDa translation product of WDV open reading frame C1-N as well as an artificially shortened 24 kDa Rep of TYLCV, cleave and join single-stranded origin DNA in vitro. Thus the pivotal origin recognition and processing activities of geminivirus Rep proteins must be mediated by the amino-terminal domain of Rep.

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