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Lecture 3 Problem: Promoter Coding Region ORF deleted protein You have cloned a new bacterial gene encoding enzyme X, sequenced the DNA, and deduced the promoter and coding region as shown above. You prepare a gene construct with the deletion above; the deletion does not remove any of the promoter nor does it alter the open reading frame. When both the control and deletion genes are expressed in E. coli and assayed for enzyme X, you find that only the control shows enzyme activity. WHY? How can you test your hypothesis? Lecture 4 Cell Culture and Viruses Reading: Chapter 4.7; 6.7; and (6.1-6.6 as review of cell biology) Molecular Biology syllabus web site 6.1 Advantages of working with cultured cells over intact organisms • More homogeneous than cells in tissues • Can control experimental conditions • Can isolate single cells to grow into a colony of genetically homogeneous clone cells 6.1 Growth of microorganisms in culture • • • • Examples: E. coli and the yeast S. cerevisiae Have rapid growth rate and simple nutritional requirements Can be grown on semisolid agar Mutant strains can be isolated by replica plating 6.1 Replica plating Plant cell culture • Production of phytochemicals naturally or by metabolic engineering with foreign DNA • Production of transgenic organisms (DNA transfer) • Clonal propagation of plants after regeneration (e.g. orchids) 6.2 Growth of animal cells in culture • Requires rich media including essential amino acids, vitamins, salts, glucose, and serum • Most grow only on special solid surfaces A single mouse cell A colony of human cells Many colonies in a petri dish 6.2 Primary cells and cell lines • Primary cell cultures are established from animal tissues • Certain types of cells are easier to culture than others • Most cells removed from an animal grow and divide for a limited period of time (about 50 doublings), then eventually die • Certain “transformed cells” may arise that are immortal and can be used to form a cell line • Transformed cells may be derived from tumors or may arise spontaneously • The rate of spontaneous transformation varies for different species 6.2 Establishment of a cell culture Figure 6-5 6.2 Some cultured cells can differentiate and form tissue-like structures Disadvantages of tissue culture • Does not always mimic in vivo conditions in terms of gene expression • Chromosome abnormalities • Cells are nondifferentiated, though some differentiated types can be grown • Primary cell cultures are short-lived and tumor cells will divide indefinitely 6.2 Cell fusion • Two different cells can be induced to fuse thereby creating a hybrid cell (heterokaryon) • Interspecific hybrids may be used for somatic-cell genetics • Certain hybrid cells (hybridomas) are used to produce monoclonal antibodies Using hybridomas for monoclonal antibody production • A mouse is injected with an antigen • B lymphocytes making antibodies begin to proliferate forming a clone of cells in the spleen or lymph nodes. • Each cell of the clone produces identical antibodies, “monoclonal antibody.” However, these B-lymphocytes are short-lived. To produce lines of indefinite growth, fusions with immortal tumor cells are required (hybridomas). • One antigen has many epitopes; multiple B lymphocyte cells produce multiple types of monoclonal antibodies (Polyclonal antibodies). To obtain monoclonal antibodies, the clonal cell lines must be separated. Using selection to produce monoclonal antibodies • Antibodies are produced by B lymphocytes, short-lived cells; each cell can produce a different antibody that recognizes a specific epitope/antigen. • Fusion with tumor cells provides continous growth of antibody-producing cell-line • The challenge is to select against non-antibody producing tumors and for antibody-producing hybridomas. • The approach is to use tumor cells that are defective in the salvage pathway for nucleotide biosynthesis (HGPRT ). Nucleotide Biosynthesis • de novo pathway (blocked by aminopterin) - • Salvage pathway (blocked in HGPRT cells) Tumor Cells • de novo pathway (blocked by aminopterin) - • Salvage pathway (blocked in HGPRT cells) Hybridomas • myeloma cells are selected as HGPRT• HGPRT- myeloma cells are fused with B-lymphocytes taken from spleen of antigen-injected mouse myeloma cells HGPRT- + B-lymphocytes HGPRT+ Plate on HAT medium: hypoxanthine, thymidine (salvage pathway) and aminopterin (blocks de novo pathway) Unfused myeloma cells die (HGPRT-): can’t use salvage pathway Unfused B lymphocytes die (primary cells with short life span) Fused hybridomas grow- each cell producing a colony secreting a unique monoclonal antibody Uses of antibodies • Immunolocalization (fluorescence, EM) • Protein purification (affinity column) • in vivo binding of antigen (therapeutic value as inhibitor or to deliver toxic drug; basic research tool) • Gene isolation (screening by expression) • Analysis of gene expression (in situ or by western analysis) 6.3 Viruses: structure, function, and uses • Viruses are small parasites that cannot reproduce by themselves • A virus infects a suitable cell and utilizes the host cell machinery to produce more viruses • A virus consists of nucleic acid (RNA or DNA) surrounded by a shell of protein • Viruses either infect prokaryotic or eukaryotic cells and the host range of most viruses is narrow • Study of viruses has furthered understanding of basic aspects of cell biology as well as the development of cancer • Have useful gene promoters for transgenic research Influenza virus virion Bacteriophage Lambda plated on E. coli Poliovirus plated on animal cells (HeLa) Budding virions of measles virus (an RNA virus)