Blumberg et al.1991. Organizer specific homeobox genes in Xenopus laevis embryos. Science 253:194196

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Blumberg et al.(1991). Organizer specific
homeobox genes in Xenopus laevis embryos. Science
253194-196

• Rationale Can genes involved in pattern
  formation, which are regionally expressed, be
  isolated by molecular means?
• Methodology Authors attempt to generate a cDNA
  library using tissue dissected from the dorsal
  lip of the blastopore (the histological
  organizer)

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A walk through the technology is it possible to
isolate transcription factors specific for
organizer region?

• Dissection accomplished from 300 blastopore lips
• Yielded approximately 80 ?g of total RNA
  (partition chromatography)
• mRNA purified from total RNA (90 ribosomal) by
  affinity chromatography

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mRNAAUUGGCUACCUÄÄÄÄÄÄÄÄÄÄÄÄ
mRNA...CGUUACGGCGCÄÄÄÄÄÄÄÄÄÄÄÄ
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• Construction of a cDNA library
• provides a snapshot of the genes expressed in
  a particular tissue
• isolate mRNA from tissue of interest (e.g.
  dorsal blastopore lip)
• convert to d.s. DNA in vitro with reverse
  transcriptase and Pol I

RNAse H
nick

• prepare ends for cloning (ligate on restriction
  sites)
• clone into vector and propagate in microorganism

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Applications of Bacterial Enzymes Synthesis and
Propagation (Cloning) of Recombinant DNA

• Purified enzymes from microorganisms can be used
  as molecular scissors to break up DNA molecules
  and piece them together
• Autonomously replicating genetic elements in
  microorganisms (e.g. plasmids) can be used to
  propagate that DNA within individual cells...

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• Restriction Endonucleases
• have a specific recognition sequence (e.g 4-6
  bp)
• many enzymes have palindromic recognition sites
• DNA cut with these enzymes is broken within the
  strand
• some of the enzymes leave a single strand
  overhang (sticky end)

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Bacterial Plasmids have been engineered to serve
as vehicles (vectors) for the replication of
foreign DNA

• Properties
• origin of replication
• restriction sites to introduce DNA
• means of selection for plasmid in bacterial cell

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Cutting of a circular plasmid with restriction
enzyme yields a linear molecule with sticky
ends...
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• Digest foreign DNA with same enzyme
• if 6 base recognition site is random in genome
  cuts every 4096 bp (46)
• human genome 3 x 109 bp7 x 105 fragments

Linearize plasmids with restriction enzyme
Entire population of transformants represents
genomic library

• single fragments are ligated to individual
  plasmid DNAs
• transformation introduces individual plasmids
  (clones) into bacteria

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Viruses can also be used to clone foreign DNA
molecules via their ability to propagate in
bacteria...
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• digest lambda DNA
• remove middle section (contains genes for
  lysogeny)
• ligate foreign DNA to lamba arms
• mix with viral coat proteins to package
  recombinant DNA

• infect E. coli

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cDNA library construction into ?-ZAP
vectorsdirectional cloning

• First-strand synthesis
• Linker-primer
• MMLV-reverse transcriptase
• Nucleotide mixture containing methylated C
• Second-strand synthesis
• second-strand dNTP mixture
• ?- 32P dATP

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Collected Fractions
(Non-denaturing Polyacrylamide Gel)
11 10 9 8 7 6 5 4
4.3 kb 2.0 kb 710 bp 600 bp 489 bp
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Ligating cDNA into the Uni-ZAP XR Vector
MCS or polylinker small region containing unique
restriction sites
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Properties of the Vector

• Accommodates DNA inserts 0-10 kb in length
• can be screened with DNA or antibody probes
• in vivo excision of the pBluescript phagemid
  possible

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DNA sequence surrounding the Multiple Cloning
Site (MCS) or polylinker
cDNA insert site
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or cDNA
Creating a genomic/cDNA library using phage
vectors...
(CLONE)
All phage plaques taken together represent
genomic library...
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How is clone of interest isolated from library?

• Information on protein sequence of interest can
  lead to the synthesis of short oligonucleotide
  probes.in this case, they were interested in
  isolating homeotic gene homologs
• Homeotic Mutations a gene, that when mutated,
  leads to a change in developmental program e.g.
  a change from one body part to another

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Homeotic Selector Genes

• Examples
• antennapedia changes antennal structures into
  leg structures (i.e. normally contributes to
  giving thoracic segments their identities
  misexpression in head leads to change in
  identity)
• ultrabithorax inactivation of the gene will lead
  to transformation of third thoracic segment into
  second segmenti.e. a fly with two sets of wings
  instead of halteres

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• Molecular cloning of genes have identified a
  common sequence element, encoding about 60 amino
  acids (homeobox), which is shared by this family
  of genes (bicoid is one example in Drosophila).
• This motif is responsible for DNA-binding
  activity, and evidence has been accumulated that
  homeobox sequences can act as transcription
  factors, regulating the expression of other genes

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The phage cDNA library was screened with a
degenerate primer encoding the homeobox region
(actually a composite of 1024 different
oligonucleotides)
Example
Screening was done using nucleic acid sequence
complementarity (nucleic acid hybridization)
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Use of Labeled DNA Probes Hybridization analysis

• The self-complementary nature of DNA can be used
  to identify its presence (and the presence of
  gene products) in cells and tissues
• DNA can be denatured to single- stranded form
• Labeled probes can then be made to reanneal
  (hybridize) to the DNA in situ...

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• Heat treatment (as shown here) or treatment with
  bases (e.g. NaOH) will cause DNA to denature.

• Under the appropriate salt and temperature
  conditions, it can be made to reanneal (base
  pair) with a complementary strand

If one DNA strand (probe) is labeled, genes can
be detected...
C/Co 1/(1 Kcot)
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• replica plate plaques or colonies onto filter
• denature DNA on filter
• incubate with radiolabeled probe
• detect cells that contain correct gene by
  autoradiography
• grow up appropriate clone

Genomic Library of bacteriophage lambda plaques
plated on petri dishes...
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• replica plate plaques or colonies onto filter
• denature DNA on filter
• incubate with radiolabeled probewash off
  unbound probe
• detect cells that contain correct gene by
  autoradiography
• grow up appropriate clone

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30 cDNA clones were identified which fell into 3
distinct classes...

• Goosecoid named due to similarity with bicoid
  gene in Drosophila. It encodes a 1.3 kb mRNA
  that is the earliest that is detectable relative
  to the other clones
• Xcad two different genes similar to the
  Drosophila gene caudal.
• Xlab closest to the fly gene labial. High
  amounts in early gastrula and also found in other
  regions of embryo

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For rest of the paper they concentrate on initial
characterizations of Goosecoid...