LT2-3 Cloning Developmental Mutations

Cloning Developmental Mutations

Locus The location on a linkage map or on a chromosome of a heritable factor
controlling a particular trait
Gene 1) Heritable factor occupying a specific locus
2) Segment of DNA that controls the production of protein
Allele Alternative forms (sequences) that can occupy a particular locus
A “wild-type” allele is the form most commonly found in a given population
Cloning 1) Making a (genetic) copy of an individual
2) Identifying the sequence of a gene for which mutation(s) have been
isolated


Cloning genes – “Identifying the sequence for a mutation”

Purpose

 Homology: Cloning allows related genes in zebrafish or other species to be
identified
 Biochemistry: Biochemical function of the gene’s protein product to be studied
 Expression/Transgenics/Bioinformatics: Look at gene’s expression during
development, analyse its regulation and effects of mis-expression using transgenic
fish

Methods

a) Location  Positional Cloning
b) Phenotype  Candidate genes

Phenotypic Analysis

 Compare phenotype with that of mutants in other
species
 Infer tissue or cell-type specific expression of gene
 Infer physical interactions with other proteins

Confirmation

 Scan sequence for non-neutral mutations
 Check expression of protein or mRNA in homozygous mutant fish
 Can injection of wild-type protein or mRNA rescue the phenotype (functional
complementation) or knockdown of mRNA by antisense RNA injection phenocopy the
mutant (can the wild-type phenocopy the mutant?)

,Phenotypic Analysis Examples

no tail (Schulte-Merker et al., 1994)

 Two no tail (ntl) mutants isolated in 1983; Mouse T gene cloned in 1990
 Ntl is the homologue of the mouse T genes – both alleles lead to truncated protein
products
 Mutant ntl die well after hatching and relatively late in development (unlike mouse
T/T embryos, die during midneurulation
and probably due to lack of allantois)
 Can therefore examine effects of
mutation in later events – ntl seems to
disturn the anterior somite patterning but floorplate and neural structures are not
affected
 Underlying defect in mesoderm development – posterior somites fail to form and
notochord fails to differentiate
 In homozygous T/T embryos posterior somites fail to form and notochord fails to
differentiate
 In 1992, putative zebrafish homologue Zf-T isolated – Zf-T amino acid sequence
69.7% identical with mouse T (high homology)
 T and Zf-T expressed in early mesoderm and in the developing notochord
 Zf-T and ntl are closely linked on chromosome 19
 Zf-T antibodies detect no protein in homozygous mutant
ntl/ntl embryos
 Coding sequence of wild-tupe and ntlb159 fish differ
(insert)
 Coding sequence of wild-type and ntlb160 fish differ
(missense)

nacre (Lister et al., 1999)

 Single nacre (nac) mutation isolated in 1999
 Pigment (melanin) normal in eyes but absent from body,
similar phenotype to cloned black-eyed white mouse
mutants
Ie. 3 candidate mouse genes: steel, c-kit, Mitf (need to
distinguish between them)
 Wild-type cells injected into nac/nac embryos generate normal pigment cells
 Wild-type cells into c-kit or Mitf mutant embryos generate normal pigment cells
(transplants suggest that nacre functions cell-autonomously)
 Wild-type cells injected into Steel mutant embryos unable to generate normal
pigment cells

, Zebrafish homologue of Mitf (microphthalmia-related protein) isolated and closely
linked to nac on chromosome 6
 Z-Mitf is expressed in developing pigment cells
 Nacre mutant carries a nonsense mutation in the coding sequence for Z-Mitf
 Injection of Z-Mitf DNA rescues phenotype
 Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-
crest derived pigment cell fate

minifin (Connors et al., 1999)

 Minifin (mfn) adults show variable loss of tail fin
 Primary defect is a partial dorsalisation of the tail bud
 Genetic regulation of dorso-ventral pattern formation is highly conserved across
many organisms
Drosophila dpp and tolloid lose the fly equivalent of vertebrate ventral pattern
elements
 2 zebrafish dpp related genes and one tolloid homologue identified
 Dpp related genes map to chromosomes 11 and 20 (Dpp eliminated from candidate)
 Zebrafish tolloid and mfn map to chromosome 1
 Tolloid related cDNA clones from all 5 alleles mfn sequenced and compared with the
wild-type zebrafish tolloid sequence
 All 5 alleles of mfn have mutations which could affect protein function – 3 are
nonsense mutations, other 2 are missense mutations affecting conserved amino
acids
 Minifin encodes zebrafish homologue of the Drosophila D-V patterning gene tolloid

sapje (Bassett et al., 2003)

 Sap mutants develop lesions in skeletal muscle where fibres detach from myosepta
 Zebrafish dystrophin gene (dmd) is associated with embryonic muscle attachment
sites
 Both dmd and sap map to chromosome 1 close to Z5508
 Homozygosity for nonsense mutation in exon 4 of dmd segregates with sap
phenotype
 Injection of dmd antisense RNA phenocopies sap




Identification of Homologous Genes

, In silico Databases: BLAST:
Genome Projects Protein
EST collections Nucleotide
Translated nucleotide
In vitro Libraries Probes
Genomic cDNA
cDNA Low stringency hybridisation
Degenerate PCR products
 New model organisms still developed for different purposes of research
(eg. shrimp for crustacean phylo geny, naked mole rat for ageing)

Show that the candidate gene is in a similar location to the target sequence

Linkage Analysis

 Linkage analysis exploits fact that the closer 2 genes are on a
chromosome the less likely it is that recombination will take
place in the intergenic region during meiosis
 Recombination creates new haplotypes (collection of specific
alleles in a cluster of tightly-linked genes on a chromosome
that are likely to be inherited together) and alleic combinations
Eg. Parental haplotypes are AB and ab
Crossovers between gene a and gene b will
generate the recombinant haplotypes Ab and
aB

Assuming we know where vestigial is (ie. need to
know the location of one of the genes) – does
purple map anywhere near vestigial?

1. Wild-type x tester stock to produce
heterozygotes
2. In heterozygotes – recombination will give an
effect (recombination in pre-cross will only
give the same combinations as they are homozygous)
3. Double heterozygotes are then backcrossed – phenotypes are easier to distinguish
4. The offspring from the backcross are then crossed together – can then see
whether pr and vg are alleles of the same gene, or are mapped closely together
 For 2 loci identified by a mutant phenotype (eg. vestigial wings and purple), to
determine the frequency of recombination events we analyse the segregation of
wild type (vg+ and pr+) and mutant (vg and pr) alleles
 When mapping new mutants – a dihybrid cross between wild-type and mutant
tester stock (“multiply mutant”)