Alan Ashworth

An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1

Members of the Rho family of small guanosine triphosphatases (GTPases) regulate the organization of the actin cytoskeleton; Rho controls the assembly of actin stress fibers and focal adhesion complexes, Rac regulates actin filament accumulation at the plasma membrane to produce lamellipodia and membrane ruffles, and Cdc42 stimulates the formation of filopodia. When microinjected into quiescent fibroblasts, Rho, Rac, and Cdc42 stimulated cell cycle progression through G1 and subsequent DNA synthesis. Furthermore, microinjection of dominant negative forms of Rac and Cdc42 or of the Rho inhibitor C3 transferase blocked serum-induced DNA synthesis. Unlike Ras, none of the Rho GTPases activated the mitogen-activated protein kinase (MAPK) cascade that contains the protein kinases c-Raf1, MEK (MAPK or ERK kinase), and ERK (extracellular signal-regulated kinase). Instead, Rac and Cdc42, but not Rho, stimulated a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun NH2-terminal kinase or stress-activated protein kinase). Rho, Rac, and Cdc42 control signal transduction pathways that are essential for cell growth.

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11‐related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domainsof the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication‐degeneration‐complementation process. Dev Den;217:279–292.

Isolation of a human cytochrome P‐450 reductase cDNA clone and localization of the corresponding gene to chromosome 7q11.2

We have isolated and sequenced cDNA clones that code for rat and human NADPH dependent cytochrome P‐450 reductase. The cDNA coding for the human protein was used to analyse, by Southern blot hybridization, DNA isolated from a panel of 8 independent humanrodent somatic cell hybrids. The results indicate that cytochrome P‐450 reductase is encoded by a single gene (POR) located on human chromosome 7(pter‐q22). Analysis of human metaphase chromosomes by hybridization in situ confirmed the results and refined the localization to 7q11.2. Northern blot hybridization revealed that in human liver the expression of the gene varies by less than 3‐fold between different individuals.

sox30: a novel zebrafish sox gene expressed in a restricted manner at the midbrain-hindbrain boundary during neurogenesis.

Abstract The Sox family of proteins is thought to act to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of sox30, a novel sox gene from the zebrafish (Danio rerio). In situ hybridization shows that sox30 is expressed in a restricted manner at the boundary between the midbrain and hindbrain during nervous system development. This expression pattern is in direct contrast to that of most other neuronally expressed Sox genes which are expressed throughout the nervous system.

Differential effect of phenobarbital and β‐naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB‐P450) by phenobarbital and its repression by β‐naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH‐cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β‐naphthoflavone.