David I. Hirsh - US grants

Experiments are proposed to elucidate the relationships between gene organization, structure and expression in the nematode, Caenorhabditis elegans. The nematode is used because of its available genetics, small genome and well described cell lineages. The genomic arrangement of the actin and collagen genes is being determined and DNA sequence analysis is in progress. The times of expression of the actin and collagen genes will be determined using the cloned sequences as hybridization probes. Expression will also bae measured by in situ hybridization in specific, well-characterized cell lineages. Experiments will be done to determine if C. elegans actin genes can be expressed and function in yeast in order to assess the conservation of the actin genes. The C. elegans actin and collagen cloned DNA's are being genetically mapped using two interbreeding strains that contain restriction site polymorphisms. More polymorphisms will be sought in other laboratory and wild strans to improve the mapping methds. Some DNA polymorphisms are due to DNA inserts and they will be studied to determine if they are transposable genetic elements. When the actin and collagen genes are mapped, mutants will be used to study the relationship between the genes and the functions of the proteins in the organism.

This Academic Research Facilities Modernization Program (ARFMP) award from the Research Facilities Office provides funds to Columbia University for the renovation and repair of the William S. Black Research Building of the College of Physicians & Surgeons, which after the modernization, will house structural studies, a multi-disciplinary research and research training program in molecular biophysics. This building was constructed in 1965 and has not previously been renovated. The ARFMP grant of $700,000 and $903,000 provided by the grantee as cost sharing will be used to modernize these research and research training facilities. It will make possible new research capabilities and collaborative interaction not possible under current conditions. This project will address the need to improve the current research infrastructure by conversion of research and research training space so as to permit consolidation of related work in structural biology and by upgrading the heating, ventilation, and air conditioning (HVAC) so that it is suitable to meet the demands of x-ray diffraction and of the computer facilities. This award contributes to the infrastructure of science by providing an improved environment for the conduct of research and for the training of quality undergraduate, graduate, and postdoctoral students and for fostering academic-industrial ties. Students trained in these laboratories will be able to serve as models for the feasibility of the training of computer-literate molecular biologists.

Trans-splicing involves the ligation of two separate RNA molecules into a single contiguous transcript. Trypanosomes, nematodes and trematodes carry out trans-splicing. In nematodes, the spliced leader RNA (SL RNA) transfers its 5'22 nucleotides from a consensus donor splice site to a consensus acceptor splice site located in the 5-untranslated region of a pre-mRNA. Consequently, some mRNAs carry a 22 nucleotide long 5'spliced leader sequence. Two proteins have been identified that bind specifically to mRNAs that contain an SL but fail to bind to mRNAs that lack an SL. The proposed research will define the detailed molecular interactions between these SL binding proteins and the SL nucleotide sequence. The methods include chemical modifications of the RNA and footprinting of the complex. Variant RNA molecules will also be selected that bind to the proteins with high affinity. These variants will help define a consensus RNA sequence and structure that will provide insight into the architecture of the molecular interactions. These studies will help establish the basic rules for RNA protein interactions. The biological function of the binding proteins and their interactions with SL on the mRNA will be studied genetically in C. elegans. The genes encoding the SL proteins will be identified. DNA sequence homologies with related genes will be sought that might relate the SL binding proteins to other RNA binding proteins of known function. The genes encoding the binding proteins will be disrupted by transposon mutagenesis. The phenotypes of those mutant nematodes lacking the binding proteins will be studied in order to define the null phenotype and to determine whether the proteins are essential. Mutations will be derived in the genes in order to elucidate their biological functions in the growth and development of C. elegans with particular attention to encoded protein domains of known functions, e.g. dominant negative phenotypes associated with nucleotide binding sites.

DESCRIPTION (Adapted from Investigator's abstract): This proposal seeks to understand the in vivo functions of endogenously expressed interleukin-1 receptor antagonist (IL-1ra). Mice lacking IL-1ra (gene knockouts) and mice over expressing IL-1ra (transgenics) will be characterized. These mutant mice to wildtype littermate controls. The proposed experiments will further analyze the underlying cellular and molecular physiological responses to the different levels of IL-1ra expression that occur in these mice. Changes in cytokine profiles will be analyzed as will leukocyte proliferation and migration. These studies will provide a deeper understanding of the in vivo mechanisms that respond to IL-1ra and provide clear phenotypes that can be used as genetic markers in a set of epistasis experiments. Double and triple mutants will be constructed involving the IL-1ra knockouts and overexpressors, IL-1 receptor nulls, IL-1beta TAbetas114 knockouts and caspase-1 (ICE) knockouts, in order to define the interactions between members of the IL-1/IL-1ra pathway. These epistasis experiments are intended to determine which phenotypes are due to the intracellular versus the secreted forms of IL-1ra, and to resolve whether another target exists for IL-1ra in addition to the IL-1 receptor, which the phenotypes of the single mutants currently strongly suggest.

The complete genome of the nematode model organism, C. elegans, has recently been published. This represents the first elucidation of the complete genome of a multicellular organism. The availability of this information is expected to open up vast new opportunities for discovery in biology. This project focuses on an essential cellular function, endocytosis, in C. elegans. This is an area of cell biology that in the past has been studied largely in yeast, in vertebrate cells in culture, and in a limited number of other model systems. The study of endocytosis (and other cellular functions) in C. elegans will ultimately allow the exploitation of not only the genomic information but also the powerful genetic tools available for studies this organism and enable important new discoveries about fundamental cellular processes and functions.

Endocytosis is the process by which cells internalize specific materials (proteins, usually) from their environment. In receptor-mediated endocytosis, an extracellular ligand first binds to its receptor on the cell surface; then, that portion of the cell membrane invaginates and ultimately pinches off to form an intracellular membrane-enclosed compartment containing the bound ligand.

A new DNAJ domain-containing gene, rme-8, was discovered by using a novel genetic screen for C. elegans mutants defective in receptor-mediated endocytosis (the rme mutants ). Rme-8 is an essential gene required for both receptor-mediated and fluid-phase endocytosis. Highly conserved homologues of RME-8 are found in plants, flies and humans, but none is present in the S. cerevisiae genome. The goal of this proposal is to study the cellular and molecular functions of rme-8 in C. elegans.

In an attempt to identify the step where RME-8 functions in endocytosis, RME-8 will be localized to an endocytic compartment by cytological co-localization with known endocytic compartment-specific markers. These subcellular localization and co-localization experiments will provide the first indication of RME-8's role in a particular step of endocytosis. The role of RME-8 in endocytosis will be also tested by examining mis-localization of known endocytic markers in rme-8 mutant strains.

In order to analyze the molecular function of RME-8, RME-8's domain structures will be studied by creating deletions of RME-8 and assaying their in vivo functions and localization. As RME-8 contains at least one known protein-protein interaction domain, namely a DnaJ domain, proteins that interact with RME-8 will be identified using the yeast two-hybrid system. These experiments will establish the important functional domains in RME-8 and identify the proteins that interact with these domains.

Rme-8 is an essential gene whose function is required in multiple cells in C. elegans. In particular, rme-8 mutants are defective in molting and in fluid-phase endocytosis. The role of RME-8 in molting as well in fluid-phase endocytosis will be studied by examining RME-8's subcellular localization in those cells associated with these mutant phenotypes, namely hypodermal cells and coelomocytes.

These experiments will define the step of endocytosis in which RME-8 functions, establish RME-8's role in that specific step, and define RME-8's possible involvement in a protein complex. RME-8 represents a previously unidentified component in endocytosis. Therefore, the discovery of RME-8's role in endocytosis might identify one of the many heretofore missing links in the endocytosis pathway. Understanding RME-8 in C. elegans will provide an important basis for understanding the functions of its close homologues in other organisms.