A window into domain amplification through Piccolo in teleost fish

I describe and characterize the extensive amplification of the zinc finger domain of Piccolo selectively in teleost fish. Piccolo and Bassoon are partially functionally redundant and play roles in regulating the pool of neurotransmitter-filled synaptic vesicles present at synapses. In mice, each protein contains two N-terminal zinc finger domains that have been implicated in interacting with synaptic vesicles. In all teleosts examined, both the Bassoon and Piccolo genes are duplicated. Both teleost bassoon genes and one piccolo gene show very similar domain structure and intron-exon organization to their mouse homologs. In contrast, in piccolo b a single exon that encodes a zinc finger domain is amplified 8 to 16 times in different teleost species. Analysis of the amplified exons suggests they were added and/or deleted from the gene as individual exons in rare events that are likely the result of unequal crossovers between homologous sequences. Surprisingly, the structure of the repeats from cod and zebrafish suggest that amplification of this exon has occurred independently multiple times in the teleost lineage. Based on the structure of the exons, I propose a model in which selection for high sequence similarity at the 5′ and 3′ ends of the exon drives amplification of the repeats and diversity in repeat length likely promotes the stability of the repeated exons by minimizing the likelihood of mispairing of adjacent repeat sequences. Further analysis of piccolo b in teleosts should provide a window through which to examine the process of domain amplification

Landscape of target: Guide homology effects on Cas9-mediated cleavage

To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleav-age. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (al-beit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in ‘seed ’ sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (posi-tions 13–18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit mod-est) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA ex-pression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9– gRNA interaction and cleavage beyond the general paradigm of site determination based on the ‘seed’ sequence and PAM

Evolution of the RNA polymerase II C-terminal domain

In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms. Originally published Proc Natl Acad Sci, Vol. 99, No. 9, Apr 200

The Caenorhabditis elegans Kinesin-3 motor UNC-104/ KIF1A is degraded upon loss of specific binding to cargo

UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P(2) through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P(2) binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P(2)in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P(2) levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P(2) binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P(2) levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P(2) binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo

Dosage compensation: X-repress yourself

AbstractDosage compensation in Caenorhabditis elegans involves the sex-specific recruitment to the X chromosome of a protein complex, the nature of which suggests that there are mechanistic links between chromosome segregation and global transcriptional regulation

Improved GAL4 and tet OFF drivers for C. elegans bipartite expression

The first generation o

Judgment

To judge, in Latin judicare, is to say the law, jus dicere, whence juris-dictio. The above sentence is a possible answer to the question: what is judging? It spells out what the word to judge says, by recalling the history from which the word originates. Why would anyone ask this question? How helpful is such an answer? Everyone knows what it is to judge. Only on the ground of such self-evidence could there be that unabating debate on the \u27 justification of particular judgments, which is the day to day business of lawyering. Only because the question can be passed over can there be controversy regarding the forms and limits of adjudication in general, a preoccupation without which jurisprudence would seem to lose its main occupation. Why ask the question? Precisely because the matter is self-evident. As soon as we examine it a little, confusion begins to set in all over. Monsieur Jourdain likely would be flattered if he knew that logicians employ the word \u27Judgment in its widest sense to designate propositions of all kinds: He has been judging all along, and therefore knows already how to do it. Even Kant follows this usage when he says that we can reduce all acts of the understanding to judgments, and goes on to propose a table that classifies all possible forms of judment into four groups of three (Kritik der reinen Vernunft, * A 67- pages of the same star editions). Could it be that almost all speaking is a saying of law? A little later in the same treatise, however, Kant restricts the sense of \u27Judgment to the act of subsuming under rules, that is, of distinguishing whether something falls under a given rule or not (casus datae legis) (id. A 132-34, B 171-74). This sense is borrowed from lawyerly usage, not from logic, for, as Kant shows, logic has nothing to say regarding this operation. There are, and there can be, no rules regarding the application of rules. If Kant is right, a sizable part of what we take to be law, and almost all jurisprudence, are nothing but a futile striving to overcome this essential unruliness of judgment. How can it be that the saying of law is lawless

Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function