Igor B. Dawid, PhD, Chief

The Laboratory of Molecular Genetics (LMG) studies regulation of gene expression and the genetic control of developmental and physiological processes in model organisms from bacteria to vertebrate animals.

Michael Cashel, who leads the Section on Molecular Regulation, has studied the mechanism of general metabolic control in bacteria; such control is mediated by a small molecule, guanosine tetra/pentaphosphate, or (p)ppGpp. Recently, the group's experiments demonstrated that (p)ppGpp is a key regulator of overall growth control in Escherichia coli. Additional experiments carried out in the past year stressed the role of the protein DksA and its relationship to (p)ppGpp in regulating the activity of RNA polymerase. It appears that DksA acts by interacting with the known transcription elongation factors GreA and GreB.

Ajay Chitnis and colleagues in the Section on Neural Developmental Dynamics are studying the formation of the nervous system in zebrafish, in particular the way in which neuronal versus non-neuronal fate is determined within the neural domain of the embryo and how different regions assume different structural and functional identities. In the past year, the group illuminated the role of genes in the zic family in regulating the formation of the hindbrain. In a related project, the group studied proteins interacting with Mindbomb (Mib); Mib is known to affect Notch signaling, which is required for proper specification of neuronal differentiation. The study revealed the protein Mosaic eyes (Moe) as a Mib-interacting protein; the group is now investigating Moe's role in modulating Notch signaling.

Robert Crouch, who heads the Section on Formation of RNA, has studied RNasesH, enzymes that degrade the RNA component of RNA/DNA hybrids. In particular, the Section has studied several aspects of bacterial, yeast, vertebrate, and retroviral RNasesH. The most important recent finding came from a collaborative study that solved the structure of the complex of a bacterial RNaseH with its RNA/DNA hybrid substrate. The structure yields important insights into the enzyme's mechanism of action and specificity and provides a basis for understanding the mechanism of substrate interactions of RNasesH from other organisms. In a closely related project, the laboratory analyzed the composition of a second enzyme, RNaseH2, from yeast, showing that it is composed of three subunits. Researchers not affiliated with the laboratory have used this information to identify the subunits of human RNaseH2 and to show the connection between mutations in these subunits and the rare genetic Aicardi-Goutieres syndrome.

Using both Xenopus and the zebrafish, Igor Dawid and colleagues in the Section on Developmental Biology employ DNA microarray techniques and mutagenesis to analyze the molecular basis of development. The laboratory characterized a mutation in zebrafish that affects a protein named BAP28. The protein plays a role in ribosome biogenesis, specifically in the processing of 18S ribosomal RNA and formation of the small ribosomal subunit. A study in Xenopus dealt with the mechanism of cell movements during gastrulation. Precise regulation of these movements is critical for formation of the embryonic body plan. The laboratory identified and characterized an enzyme that activates the small GTPase Rho. Rho is known to be an essential factor that regulates the cellular cytoskeleton and is important for regulation of cell movements in the embryo.

Judith Kassis, who heads the Section on Gene Expression, is studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila and the nature of DNA elements responsible for silencing. Recent work showed that several proteins, most notably an Sp1/Klf-type protein and the Pho and Pho-like proteins, are required for functional silencing through sequences called Polycomb Response Elements (PREs). The laboratory has characterized in some detail the sequences responsible for Sp1/KLF binding in a particular PRE. Investigators studied recruitment of the silencing protein complex to the PRE, showing that Pho and Pho-like play largely redundant functions in this process. In addition, studies analyzed the transcriptional regulation of the Engrailed gene and the role played by the Engrailed PRE in this process.

Jim Kennison, who heads the Section on Drosophila Gene Regulation, studies mechanisms of transcriptional regulation in Drosophila, with a focus on transacting regulators of homeotic genes. Genetic screens have led Kennison to the isolation of a series of regulatory genes that play fundamental roles in controlling the expression of homeotic genes. He has now characterized several proteins encoded by previously identified genes and recently found and is investigating the nature of additional genes that regulate the known homeotic gene Sex combs reduced. The group has studied nuclear import and export of the homeodomain protein Prospero. Caliban, a protein whose human homologue has been implicated in colon and lung cancer, controls such import/export activity. In a related project, investigators defined cis-acting regulatory sequences in the Sex combs reduced gene, leading to the interesting conclusion that two genetic elements about 70 kb apart in the Sex combs reduced gene must be in cis to maintain proper repression.

Judith Levin, who heads the Section on Viral Gene Regulation, and her colleagues study the life cycle and mechanism of reverse transcription in HIV, particularly the role of nucleocapsid protein in HIV-1 replication. Nucleocapsid is a chaperone that is required for destabilization of a stem-loop structure during viral replication. Members of the Section elucidated structural requirements for this reaction in the protein and its nucleic acid partners. A distinct project concerns the mechanism of action of the cytidine deaminase APOBEC3G. The enzyme can inhibit viral replication in the absence of the viral protein Vif. The group was able to purify functional APOBEC3G and thus study its physical and functional properties. Structure-function studies on the purified protein led to experiments that defined the role of the APOBEC3G zinc fingers in the protein's function.

Tom Sargent and his colleagues in the Section on Vertebrate Development study neural crest specification in Xenopus. The neural crest is the source of a large variety of cell types in the development of vertebrate embryos. The researchers identified three targets of the transcription factor AP-2, an important regulator of neural crest development. The first, the INCA protein, has been shown to play a major role in neural crest development. To illuminate INCA function, the investigators used the yeast two-hybrid method to identify proteins interacting with INCA. The major interacting protein is p21-activated kinase (PAK4); its putative role in neural crest development is now under study. The second Ap-2 target gene encodes PCNS, a protocadherin that is expressed in the neural crest and the somites. PCNS has been shown to be important for neural crest cell migration and for the formation of properly structured somites. The third target protein under study is Myosin10, a member of the family of non-muscle myosins. Results to date indicate that Myosin10 is required for craniofacial cartilage development.

Brant Weinstein and colleagues in the Unit on Vertebrate Organogenesis have expanded their work on the formation of the vascular system in the zebrafish. They achieved two major advances this year. First, given that earlier studies were limited to observations of cells in culture systems, researchers were unable to understand how tubular blood vessels form from vascular endothelial cells. However, the optical clarity of the zebrafish embryo allowed sophisticated high-resolution time-lapse fluorescence imaging of live transgenic zebrafish. Based on direct observations of the assembly of blood vessel tubes from endothelial cells, Weinstein and colleagues provided support for a model in which the formation and fusion of vacuoles within endothelial cells drive vascular tube formation. The second advance concerns the lymphatic system, an important second vascular system that has been difficult to study in other vertebrate model systems. The group demonstrated, for the first time, the existence of a lymphatic vascular system in the zebrafish and provided its general anatomical and functional characterization. The experiments also yielded unequivocal, in vivo evidence for the origin of lymphatic endothelial cells from primitive veins. These findings open up the opportunity for further study of the lymphatic system by genetic, molecular, and developmental approaches.

Robert Weisberg and his colleagues in the Section on Microbial Genetics study the mechanism of antitermination in bacteriophages, a process that controls gene expression at the stage of termination of transcription. They showed that antitermination depends on a specific structure of the nascent RNA that interacts with the elongating RNA polymerase. In a separate project, they determined the genome sequence of bacteriophage B40-8 and are now annotating it. The host of B40-8 is Bacteroides fragilis, a human commensal bacterium that is frequently pathogenic. The sequence showed that B40-8 is a very distant relative of all known bacterial viruses. In view of the dissimilarity of B40-8 to known organisms, the analysis may reveal hitherto unknown mechanisms of transcriptional control.

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