Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.     

A structural change in the kinesin motor protein that drives motility

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.     

Functional recognition of the 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.     

pHi regulatory ion transporters: an update on structure, regulation and cell function

Intracellular pH (pH_i) is a major regulator of various and critical cellular functions. A close regulation of pH_i is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H⁺ or equivalent H⁺ into and out of the cell. Besides pH_i, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters. Received 30 December 1998; received after revision 4 February 1999; accepted 9 February 1999