Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins.

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.     

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.     

A novel POU domain protein which binds to the T-cell receptor beta enhancer.

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.     

Improved tests for heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

The neutral theory of molecular evolution predicts that the ratio of polymorphisms to fixed differences should be fairly uniform across a region of DNA sequence. Significant heterogeneity in this ratio can indicate the effects of balancing selection, selective sweeps, mildly deleterious mutations, or background selection. Comparing an observed heterogeneity statistic with simulations of the heterogeneity resulting from random phylogenetic and sampling variation provides a test of the statistical significance of the observed pattern. When simulated data sets containing heterogeneity in the polymorphism-to-divergence ratio are examined, different statistics are most powerful for detecting different patterns of heterogeneity. The number of runs is most powerful for detecting patterns containing several peaks of polymorphism; the Kolmogorov-Smirnov statistic is most powerful for detecting patterns in which one end of the gene has high polymorphism and the other end has low polymorphism; and a newly developed statistic, the mean sliding G statistic, is most powerful for detecting patterns containing one or two peaks of polymorphism with reduced polymorphism on either side. Nine out of 27 genes from the Drosophila melanogaster subgroup exhibit heterogeneity that is significant under at least one of these three tests, with five of the nine remaining significant after a correction for multiple comparisons, suggesting that detectable evidence for the effects of some kind of selection is fairly common.      

Neurogenetic interactions and aberrant behavioral co-morbidity of attention deficit hyperactivity disorder (ADHD): dispelling myths

Background

Attention Deficit Hyperactivity Disorder, commonly referred to as ADHD, is a common, complex, predominately genetic but highly treatable disorder, which in its more severe form has such a profound effect on brain function that every aspect of the life of an affected individual may be permanently compromised. Despite the broad base of scientific investigation over the past 50 years supporting this statement, there are still many misconceptions about ADHD. These include believing the disorder does not exist, that all children have symptoms of ADHD, that if it does exist it is grossly over-diagnosed and over-treated, and that the treatment is dangerous and leads to a propensity to drug addiction. Since most misconceptions contain elements of truth, where does the reality lie?

Results

We have reviewed the literature to evaluate some of the claims and counter-claims. The evidence suggests that ADHD is primarily a polygenic disorder involving at least 50 genes, including those encoding enzymes of neurotransmitter metabolism, neurotransmitter transporters and receptors. Because of its polygenic nature, ADHD is often accompanied by other behavioral abnormalities. It is present in adults as well as children, but in itself it does not necessarily impair function in adult life; associated disorders, however, may do so. A range of treatment options is reviewed and the mechanisms responsible for the efficacy of standard drug treatments are considered.

Conclusion

The genes so far implicated in ADHD account for only part of the total picture. Identification of the remaining genes and characterization of their interactions is likely to establish ADHD firmly as a biological disorder and to lead to better methods of diagnosis and treatment.

     

Effects of intima stiffness and plaque morphology on peak cap stress

Background  

Rupture of the cap of a vulnerable plaque present in a coronary vessel may cause myocardial infarction and death. Cap rupture occurs when the peak cap stress exceeds the cap strength. The mechanical stress within a cap depends on the plaque morphology and the material characteristics of the plaque components. A parametric study was conducted to assess the effect of intima stiffness and plaque morphology on peak cap stress.