1 1 Institute of Biology University of Iceland October, 2005
Lecture outline The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families
The phenotype Lecture outline The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine The phenotype of an organisms is a the product of an interacting process in the development of the organism. The main factors of that interaction are genes environment the individual itself (whatever it has become at any one moment) None of these factors can alone determines the phenotype instead their interaction does
The method of approach The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine The method of approach to understand the phenotype is to perturb the process, to poke the system, and attempt to define the unitary steps of the process. Can perturb any of the interacting factors: genes, environment, or individual. Perturbations of environment and of individual (e.g. perturb intracellular environment of a subprocess at a particular moment in development). A genetic perturbation by mutation may be easiest. It can be replicated, stored and studied in connection with other perturbations. Warning: Leads to a geno-centric view that genes determine phenotype. Remember: genes, environment and individual interact to produce phenotype.
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine A letter to Max Perutz from Sidney Brenner 5 June, 1963 Dear Max, These notes record and extend our discussions on the possible expansion of research activities in the Molecular Biology Laboratory.... It seems to me that, both in development and in the nervous system, one of the serious problems is our inability to define unitary steps of any given process. Molecular biology succeeded in its analysis of genetic mechanisms partly because geneticists had generated the idea of one gene one enzyme, and the apparently complicated expressions of genes in terms of eye color, wing length and so on could be reduced to simple units which were capable of being analyzed....
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine The experimental approach that I would like to follow is to attempt to define the unitary steps in development using the techniques of genetic analysis.... Another possibility is to study the control of flagellation and ciliation. This again is a differentiation in higher cells and its control must resemble the control in amoebo-flagellates.... As a more long term possibility, I would like to tame a small metazoan organism to study development directly. My ideas on this are still fluid and I cannot specify this in greater detail at the present time.
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine Proposal to Medical Research Council, October 1963 The new major problem in molecular biology is the genetics and biochemistry of control mechanisms in cellular development. We propose to start work in this field and gradually make it the Division s main research.... Part of the success of molecular genetics was due to the use of extremely simple organism which could be handled in large numbers: bacteria and bacterial viruses.... We would like to attack the problem of cellular development in a similar fashion, choosing the simplest possible differentiated organism and subjecting it to the analytical methods of microbial genetics....
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine Thus we want a multicellular organism which has a short life cycle, can be easily cultivated, and is small enough to be handled in large numbers, like a micro-organism. It should have relatively few cells, so that exhaustive studies of lineage and patterns can be made, and should be amenable to genetic analysis.... We think we have a good candidate in the form of a small nematode worm, Ceanorhabditis briggsiae, which has the following properties. It is a self-fertilizing hermaphrodite, and sexual propagation is therefore independent of population size. Males are also found (0.1%), which can fertilize the hermaphrodites, allowing stocks to be constructed by genetic crosses. Each worm lays up to 200 eggs which hatch in buffer in twelve hours, producing larvae 80 µ in length. These larvae grow to a length of 1 mm in three and a half days, and reach sexual maturity. However, there is no increase in cell number, only cell mass. The number of nuclei becomes
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine constant at a late stage of development, and divisions only occur in the germ line. Although the total number of cells is only about a thousand, the organism is differentiated and has an epidermis, intestine, excretory system, nerve and muscle cells. Reports in the literature describe the approximate number of cells as follows: 200 cells in the gut, 200 epidermal cells, 60 muscle cells, 200 nerve cells. The organism normally feeds on bacteria, but can also be grown in large quantities in liver extract broth.... To start with we propose to identify every cell in the worm and trace lineages. We shall investigate the constancy of development and study its genetic control by looking for mutants. Later Ceanorhabditis elegans was chosen instead.
The problem of phenotype Dear Max Sidney Brenner A Nobel Prize in Medicine The 2002 Nobel Prize in Medicine Given to three C. elegans researchers for their discoveries concerning genetic regulation of organ development and programmed cell death Sidney Brenner John Sulston Robert Horvitz
vs molecular biology An observed pattern. Is it a real phenomenon or crud in your pipette? Answer: Mendelize. Use the rules of genetics to dissect the problem. How do we find an answer to this problem? Sequence the genome? Do the genetics?
and molecular biology Use genetics and molecular biology in conjunction. Use genetics to cut down on the molecular biology. Don t sequence the whole genome of every organism you encounter. Instead, do genetics.
Androdioecious mating system in C. elegans Male and self-fertilizing hermaphrodite. Six linkage groups or chromosomes, I, II, III, IV, V and X. Spontaneous non-disjunction of X produces O gametes which on fertilization give XO which develop into male. Certain strains exist which produce 20 40% males. Mutation in fog-2 gene (feminization of gonad) feminizes hermaphrodite gonad turning it into a female. Thus a fully sexual system is possible.
Selfing and crossing at will, storing Selfing in hermaphrodites. 1:2:1 segregation of mutation by Mendelian rules. Pure lines Generation of (a large number of) affected individuals homozygous for a recessive mutation Use male to cross a mutation into a strain Freeze worms at 80 C and rethaw
C. elegans genetic nomenclature Genes have names with three italicized letters, a hyphen and an Arabic numeral: dpy-5, let-37, unc-11, egl-2. Names refer to mutant phenotype originally discovered and/or most easily scored: dumpy for dpy, lethal for let, uncoordinated for unc. Mutations are given names with one or two italicized letters and a numeral, e.g. e61 or mn138. The letter refers to a specific laboratory which found or generated the mutation. dpy-5(e61), let-37(mn138) Suffix can be attached to mutation name, for example e61sd, where sd stands for semi-dominant phenotype. Wild type often designated with (+) Heterozygotes designated as: dpy-5(e61) unc-13(+) / dpy-5(+) unc-13(e51)
A strain is a set of individuals of a particular genotype capable of producing more individuals of the same genotype. Strains designated with two non-italicized upper case letters identifying laboratory and a number, e.g. RW4 An open community of researchers which shares information and strains. The ideas of keeping information in the public domain, for example by releasing genome sequence immediately into the public domain, are very strong in the C. elegans community.
C. elegans life cycle
C. elegans life stages
Perturbations, mutational, environmental, individual powerful techniques Phenotype is due to interaction of genes, environment, and current state of the individual. To study, must perturb system: Genetic perturbation. Generate mutation easily, for example with EMS. Because of diploid nature deleterious recessive mutations can be made, kept in heterozygous state and made homozygous for study as need be Environmental perturbation. RNAi. A potent and specific intracellular environmental interference of gene action by double-stranded RNA. Three main methods: RNAi by injection, by soaking, and by feeding. RNAi is systemic in the worm. RNAi is heritable (for a few generations) and can be passed from one generation to the next with a single sperm.
Perturbation of individual or perturbation of the current state of the system. Laser ablation of specific cells. Worm development can be followed live under the microscope. Specific cells can be killed by shooting a laser at them. Mount a life worm in agar, with some bacteria to keep it happy. Watch development and cell division under microscope. Kill specific cells with laser ablation.
Genetic analysis Lecture outline Complementation is an important first step in mutational analysis of any trait. Testing sets of mutations to see if they are the same or different. What is the genetic complexity of the trait? Once a mutation is known for some trait modifiers can be searched for to reveal gene interaction: Genetic suppression. Very large numbers of individuals can be grown and powerful selection applied. Because worm grows as a self-fertilizing diploid both dominant and recessive suppressors can be recovered Intragenic and extragenic. Intragenic suppression include: same site replacement, compensatory mutation, alteration of splicing... Extragenic suppression include: alterations of splicing, translation decay, bypass, dosage effects...
Genetic enhancers. A mutation in one gene that intensifies the phenotype caused by a mutation in another gene. Dominant enhancers. For example intergenic noncomplementation: mutations in two genes fully recessive to their wild type form which fail to complement. Double heterozygote still shows mutant phenotype. Recessive enhancers. Screens for recessive enhancers of partial loss-of-function mutations can be designed to identify additional components acting in the same pathway. Genetic mosaics. Different cells in an individual have different genotype. Mostly in C. elegans made by having an individual homozygous for a recessive mutation and carrying an extra-chromosomal wild type allele in certain cells.
The vulva Lecture outline The C. elegans hermaphrodite vulva develops during post-embryonic development from ventral epidermal precursor cells, and connects the developing uterus to the external environment. In the adult the vulva is necessary for egg-laying and for copulation with males. Three main reasons why the vulva has been studied First, it serves as a paradigm for organogenesis. In particular, vulva development represents a well-understood case in which invariant development arises from multiple cell-cell interactions. It is also a striking example of tissue remodeling: the formation of a hole at a precise location in an organism. Second, it has been important for the genetic analysis of signaling and signal transduction by epidermal growth factor (EGF)-receptor LET-23 and RAS LET-60
Third, it has become a paradigm for examining the interactions among regulatory pathways, notably the antagonism of EGF-receptor (LET-23) and Notch (LIN-12) pathways.
Vulval development is a multi-step process
The vulva, overview
The vulva, view of vulval lineages
The vulva, example defects in induction
The vulva, major signaling pathways
First metazoan, second eukaryote Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families C. elegans was the first metazoan and only the second eukaryote after Saccharomyces cerevisiae to have its genome completely sequenced. Sequencing is finished down to the last nucleotide. Holocentric chromosomes (no centric heterochromatin) allow sequence from telomere to telomere Genome size about 100 Mb. 20 E. coli. 1/30 of human.
Some bioinformatics tools Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families WormBase www.wormbase.org Textspresso at WormBase WormBook textttwww.wormbook.org WormAtlas textttwww.wormatlas.org CGC, Caenorhabditis Center texttthttp://biosci.umn.edu/cgc/cgchomepage.htm
Gene structure Lecture outline Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families 22,227 protein-coding gene, including 2,575 alternatively-spliced forms. 126,477 predicted unique, coding exons, which account for 25.55% of the genome Relatively small genes, 3 Kb Median size of exons is 123 bp 106,909 predicted unique introns modal size for introns is 47 bp; largest confirmed intron is 21,230 bp
Noncoding RNA genes Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families approximately 1300 genes that produce functional noncoding RNA (ncrna) 569 nuclear and 22 mitochondrial transfer RNA (trna) genes. Also 1072 probable trna pseudogenes. 275 ribosomal RNA (rrna) genes. 18S, 5.8S, and 26S from a 7.2 Kb 55 tandem repeat on I. 5S from a 1Kb 110 tandem repeat on V 140 trans-spliced leader RNA genes. Approximately 70% of mrnas are covalently modified at their 5 end by the addition of 22-nt trans-spliced leader RNA sequences.
Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families 120 microrna (mirna) genes. Genomically-encoded, untranslated RNA of 20 25 nt. lin-4 and let-7 the first two known mirnas, govern temporal aspects of development and down-regulate gene expression by interacting with partially complementary sequences in the 3 UTRs of their target genes. Control such diverse events as development, metabolism, cell fate and cell death. 30 small nucleolar RNA (snorna) genes. Incompletely characterized at present 70 spliceosomal RNA genes. Some are found in several copies dispersed in the genome (e.g. 11 U1, 12 U2, 6 U4, 9 U5, and 10 U6).
Protein-coding gene families Genome sequence Some tools Gene structure Genomic classification of protein-coding gene families How do we know that the genome contains 22,227 protein-coding gene, including 2,575 alternatively-spliced forms? Similarity, homology, and shared functions. Orthology and paralogy. Duplications and gene loss. Bioinformatics tools, BLAST, HMM, HMMER... Phylogenetic classification Functional classification
In about 40 years the worm Ceanorhabditis elegans has become established as a major model organisms for studying development. From humble beginnings in 1963 to a Nobel Prize in Medicine in 2002. Androdioecious mating system. Mutation. Mendelian genetics, generation of large numbers of affected individuals. Freezing strains. Short life cycle. Transparent. 959 cells, constant cell lineage. 1090 cells, 131 die by apoptosis. Perturbations, mutational, environmental, individual
Lecture outline Genetic analysis. Complementation tests. Modifier screens. Genetic suppression. Genetic enhancement. Genetic mosaics. The vulva as an example of genetic dissection of organogenesis.. C. elegans first metazoan and second eukaryote after Saccharomyces cerevisiae to have its genome completely sequenced. Important bioinformatics tools www.wormbase.org Examples of gene structure. coding and non-coding genes. Genomic classification of protein-coding gene families.