High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.     

On the genomic location of the exuperantia1 gene in Drosophila miranda: the limits of in situ hybridization experiments

In situ hybridization to Drosophila polytene chromosomes is a powerful tool for determining the chromosomal location of genes. Using in situ hybridization experiments, Yi and Charlesworth recently reported the transposition of the exuperantia1 gene (exu1) from a neo-sex chromosome to the ancestral X chromosome of Drosophila miranda, close to exuperantia2 (exu2). By characterizing sequences flanking exu1, however, we found the position of exu1 to be conserved on the neo-sex chromosome. Further, the exu2 gene was found to be tandemly duplicated on the X chromosome of D. miranda. The misleading hybridization signal of exu1 may be caused by multiple copies of exu2, which interfere with the hybridization of the exu1 probe to its genomic position on the neo-X chromosome. This suggests that flanking DNA should be used to confirm the positions of members of gene families.     

Heterochromatic self-association, a determinant of nuclear organization, does not require sequence homology in Drosophila

Chromosomes of higher eukaryotes contain blocks of heterochromatin that can associate with each other in the interphase nucleus. A well-studied example of heterochromatic interaction is the brown(Dominant) (bwD) chromosome of D. melanogaster, which contains an approximately 1.6-Mbp insertion of AAGAG repeats near the distal tip of chromosome 2. This insertion causes association of the tip with the centric heterochromatin of chromosome 2 (2h), which contains megabases of AAGAG repeats. Here we describe an example, other than bwD, in which distally translocated heterochromatin associates with centric heterochromatin. Additionally, we show that when a translocation places bwD on a different chromosome, bwD tends to associate with the centric heterochromatin of this chromosome, even when the chromosome contains a small fraction of the sequence homology present elsewhere. To further test the importance of sequence homology in these interactions, we used interspecific mating to introgress the bwD allele from D. melanogaster into D. simulans, which lacks the AAGAG on the autosomes. We find that D. simulans bwD associates with 2h, which lacks the AAGAG sequence, while it does not associate with the AAGAG containing X chromosome heterochromatin. Our results show that intranuclear association of separate heterochromatic blocks does not require that they contain the same sequence.     

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, approximately 5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.     

Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.     

A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1-M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met(823)) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC(50) for glycine or the peak NMDA EC(50) but decreased the steady-state NMDA EC(50) and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC(50), and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met(823) in regulation of NMDA receptor function.     

Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells

Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilized cells inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists in this system. Nuclear actin was localized outside the ethidium bromide-stained region, in the extrachromosomal nuclear domain. In permeabilized cells, the appearance of nuclear actin and cofilin was not significantly affected by increasing [Ca(2+)] and/or adding guanosine 5'-O-(3-thiotriphosphate), but was greatly promoted when ATP was withdrawn. Similarly, ATP depletion in intact cells also induced nuclear actin accumulation. In contrast to the effects of latrunculin B, ATP depletion was associated with an increase in cortical F-actin. Our results suggest that the presence of actin in the nucleus may be required for certain stress-induced responses and that cofilin is essential for the nuclear import of actin.     

Membrane topology of a metabotropic glutamate receptor

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.     

X-ray structure of a novel matrix metalloproteinase inhibitor complexed to stromelysin

A new class of matrix metalloproteinase (MMP) inhibitors has been identified by screening a collection of compounds against stromelysin. The inhibitors, 2,4,6-pyrimidine triones, have proven to be potent inhibitors of gelatinases A and B. An X-ray crystal structure of one representative compound bound to the catalytic domain of stromelysin shows that the compounds bind at the active site and ligand the active-site zinc. The pyrimidine triones mimic substrates in forming hydrogen bonds to key residues in the active site, and provide opportunities for placing appropriately chosen groups into the S1' specificity pocket of MMPS: A number of compounds have been synthesized and assayed against stromelysin, and the variations in potency are explained in terms of the binding mode revealed in the X-ray crystal structure.