Download MICRO-MANIPULATION OF CHICKEN CHROM OSOMES AND

MICRO-MANIPULATION
OF CHICKEN CHROMOSOMES
AND DEVELOPMENT
OF CHROMOSOME-SPECIFIC
DNA LIBRARIES.
F. Abel Ponce de Leon 1, Yukui Li 1, Sakthikumar
Ambady 1, David Burke 1, James
Bitgood 2 and James Robl 1.
1Dept. of Veterinary
MA. 01003-6410.
and Animal
Sciences, University
of Massachusetts,
Amherst,
2Dept. of Poultry Sciences, University of Wisconsin, Madison, WI 53706
INTRODUCTION
The genome of Gallus domesticus is about 1/3 of the mammalian genome, or
approximately 1.1 to 1.4 pg of DNA per haploid cell. The chicken chromosome
complement is divided in two distinct groups of 10 macrochromosomes
and 29
microchromosomes, for a total of 39 chromosomes per haploid genome. This translates
to having about 65 % of the genome contained in the macrochromosomes and 35 % in
the microchromosomes.
Chromosome 1 contains about 17 % of the total DNA, while
the Z chromosome contains about 8 % (Bitgood and Shoffner, 1990; Fechheimer,
1990). This cytogenetic observation correlates well with the fact that approximately 90
% of all genes, so far mapped, have been assigned to macrochromosomes (Bitgood and
Somes, 1990).
The first chicken linkage map was published by Hutt (1936) and contained 18
loci distributed in five linkage groups. At present the map consists of over 200 loci,
and over 20 linkage groups and some gene assignments to microchromosomes
(Levin et al. 1994). During the last 5 decades the development of the chicken map
has been approached mainly by family segregation studies and linkage anchoring
based on chromosomal rearrangement
break points. Other approaches such as
somatic cell hybrids (Kao, 1973) and chromosomal isotopic in situ hybridization
have also been utilized but have not yet made significant contributions to the
chicken genome map.
Other approaches have been implemented with the objective to produce a 10
cM chicken gene map within the next five to ten years. These include the
development
of two reference mapping populations
(Bumstead et al , 1992,
Crittenden et al, 1993). These populations are enabling the construction of genetic '
linkage maps based on molecular genetic markers. Likewise, the utilization of
chromosomal fluorescent in-situ hybridization (FISH) is providing information
about anchor loci to facilitate chromosomal assignments of all available linkage
groups (Ponce de Leon et al., 1992), hence allowing the development of cloning
strategies for important regions of the genome. FISH will help in developing a map
of regularly spaced polymorphic loci by providing quick and accurate localization of
polymorphic genomic sequences.
Partitioning of the chicken genome into small units to concentrate mapping
efforts in small regions of the genome becomes an important task, particularly when
over 65 % or the genome is compartmentalized
into very few chromosomes.
Fragmenting
the genome by individual
chromosomes
and construction
of
chromosome-specific
DNA libraries is therefore a logical approach. We have
microisolated and microcloned two chicken macrochromosomes.
Our work was
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facilitated by the fact that chicken macrochromosomes are identified by morphology
which allowed the microisolation of 10 copies of the chromosome of interest. This
small number of copies yielded about 0.7 to 1.0 pg of DNA as the starting cloning
material. We describe here the general strategy for chromosome microisolation and
microcloning, as well as the use of the strategy for the identification and FISH
assignment of large insert sequences.
MICROCLONING
STRATEGY
Metaphase preparations. Conventional fibroblast cell cultures were carried
out as previously described (Ponce de Leon et al. 1992). Briefly, after the hypotonic
treatment of the cell suspension, chromosomes are fixed by three changes (5 min,
each) of methanol:acetic acid at 9:1, 5:1, and 3:1 before placing the cells on cleaned
ice cold coverslips. This mild fixation reduces depurination of DNA caused by
prolonged exposure to 3:1 methanol:acetic acid fixation as it is done with
conventional procedures.
Microscraping. With the help of an inverted microscope and hydraulic micro
manipulator, chromosomes are scraped from the surface of coverslips. After
scraping is completed the scraped chromosome is picked up with a micro needle and
transported to a siliconized coverslip. For each experiment ten copies of the
chromosome of interest are accumulated.
Sau3A I Adaptor. Two oligomers, a 28mer and a homologous 24mer
designed to include a Sau3A I overhang site at its 5' end and an EcoR I restriction
site close to the 3' end were prepared. Phosphorilation of the 28mer at its 5' end and
annealing of the two oligomers to form the double stranded adaptor molecule have
been previously described (Ponce de Leon and Robl, 1992). This adaptor was
designed to be ligated to chromosomal inserts generated by the microcloning
procedure. The adaptors provide a known priming site for polymerase chain
reaction (PCR) amplification of chromosomal inserts (Fig 1).
Microcloning. General procedures and buffer compositions have been
described elsewhere (Ponce de Leon and Robl, 1992; Saunders et al. 1989). Briefly,
scraped chromosomes are covered with a 1 nl drop of proteinase K buffer that is
immediately protected from evaporation with a large drop of heavy mineral oil.
Protein digestion is carried out for 2 hours at 37 C. The coverslip carrying the
chromosomal DNA is inverted and placed in an oil chamber that is filled with heavy
mineral oil. The chromosomal DNA remains in the 1 nl hanging drop. Three phenol
and one chloroform:isoamylalcohol (24:1) extractions are followed by a Sau3A I
(80U/ul! nuclease digestion. The preparation is incubated for 2 hours at 37C and the
enzyme is inactivated at 65C for 30 min. Sau3A I adaptors, T4 ligase buffer and T4
ligase are added to the nanoliter drop and incubated overnight at 4C. The hanging
drop is then taken in a micropipette and transfered to a 10 ul volume of Bgl II
digestion reaction to digest the adaptor dimer molecules generated after ligation.
After inactivation of the Bgl II enzyme the preparation is transferred to a 100 ul
PCR reaction. This reaction includes 1 uM primer (24mer oligomer). Amplification
is checked by electrophoresis in a 2% agarose gel.
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CHROMOSOME
PAINTING.
Fluorescent
in situ hybridization
(FISH).
The protocol as outlined by
Lichter et al., (1988, 1990) and Ponce de Leon et al., (1992) was carried out. Slides
containing metaphase spreads were incubated for exactly two minutes, in 70%
deionized formamide,
2X Sodium saline citrate (2X SSC) at 70°C. Slides were
immediately dehydrated in 70%, 90%, 100% ice cold ethanol for five minutes each.
The pool of amplified chromosomal inserts (chromosome
cocktail) was
labeled by nick translation using biotin-16-dUTP which substitutes dTTP in the
standard nick translation reaction mixture. Since genomic DNA contains repetitive
sequences that are common to other chromosomes, we prepared competitor DNA in
order to prevent hybridization signals originating from repetitive sequences. For this
chicken genomic DNA obtained from fibroblasts or fetal tissue and salmon sperm
DNA were digested with DNAse I to produce fragments with equivalent size
distribution as the nick translated DNA probe.
_
Between 30 and 60 ng of labeled probe DNA were mixed with 2 to 4 ug. of
competitor chicken DNA and enough salmon sperm DNA to yield a total of 10 ug of
DNA in 10 ul of hybridization solution. The DNA mixture was denatured at 75°C
for 5 minutes, followed by incubation at 37°C for 10 to 15 min to allow preannealing
of repetitive DNA sequences. The denatured and preannealed DNA mixture was
then applied to prewarmed (42°C) slides with denatured chromosome preparations.
Slides were placed in a humidified chamber and incubated overnight at 37uC.
Following the hybridization
procedure,
slides were washed in 50%
formamide (3 times for 5 min at 420C) and in 0.1X SSC (3 times for 5 min at 60°C).
Slides were then incubated in blocking solution (3% BSA, 4X SSC) for 30 rain at
37°C. Detection of the probe was accomplished by incubating the slides, in the dark,
for 30 min at 37°C with fluorescein isothiocyanate (FITC)-conjugated
avidin DCS
(5 ug/ml, Vector Laboratories) which is made up in 4X SSC, 0.1% Tween 20, 1%
BSA. After incubation, the excess detection solution was washed (in the dark) in
4X SSC, 0.1% Tween 20, 3 times for 5 min at 42°C. Slides were counterstained in a
200 ng/ml propidium iodide solution. After washing, slides were mounted in
antifade p-phenylenediamine
free base (Sigma) solution (PPD-11).
Microscopy.
Slides were screened under a microscope (Zeiss, Axioskop)
equipped for epifluorescence
microscopy.
Propidium iodide staining and FITC
FISH signals will be detected with 546/590 and 450/520 excitation/barrier
filter
combinations,
respectively.
A SIT 66 video camera, that is attached to the
microscope, was used to capture images that were digitized by an Image 1/AT
software (Universal Imaging Inc.). Images were stored in 44 mb disk cartridges
(Bernoulli). Photomicrographs of digitized images were prepared with a color video
printer (Sony, model UP 5000).
CHROMOSOME
SPECIFIC
DNA LIBRARIES.
A fraction of the amplified chromosome specific inserts was digested with
Sau 3A I according to the manufacturer' instructions. This generated Sau 3A I sticky
ends that were ligated to a comparable site of the Bam HI Lambda Zap II vector
(Stratagene). After infection of NM554 cells and plating recombinant clones can be
identified by white/blue screening. Libraries have been amplified and stored as
described in the manufacturer's
protocols. PCR amplification
of individual
2O
chromosomal inserts can be done using the RNA polymerase
flanking sites of the vector.
CHROMOSOME
T3 and T7 priming
1.
A chromosome 1 library of small inserts has been generated and is being used
for identification of clones containing microsatellite sequences. The chromosome
cocktail has been used, both as a painting probe and as a probe for identification of
chromosome 1 cqspaid clones. This latter use of the chromosome cocktail was
accomplished by "_':P end labeling of the chromosome 1 cocktail and blocking of
repetitsve DNA sequences by annealing of Cot 1 unlabeled chicken DNA (Fig 2).
Therefore, unique over represented sequences and/or middle repetitive sequences
were used for cosmid screening. One hundred positive cosrnid clones were isolated,
rescreened, and FISH assigned in pools of five per experiment. Seventy-two cosmid
clones were chromosome 1 positive and 20 of these clones have been assigned to
specific sites on chromosome 1 (Fig 3).
Z CHROMOSOME.
The Z chromosome has been microcloned as described before. We have use
the Z chromosome cocktail as a painting probe to asses its origin and purity. This is
important because contamination with DNA Originating from other chromosomes
could compromise
the purity of the preparation.
Contamination
results from
erroneous
chromosome
identification
before the scraping and/or
accidental
scraping of pieces of other chromosomes surrounding the chromosome
being
scraped.
The Z chromosome painting probe has been used to analyzed the NM 7659
t(Z;1) chromosome
rearrangement
(Zartman,
1973). Preliminary observations
resulting from the analysis of the FISH signals obtained with the Z chromosome
cocktail indicate that the break points for the reciprocal translocation occurred at
Zp23 and lq13. The Zp24 band has been translocated to lqll. This would indicate
the translocated lq lost band lq12. This loss of chromatin might be one of the
reasons or the most important reason why birds homozygous for the translocation
have not been generated
in spite of successive efforts (Bitgood, personal
observation). Our intention is to use the NM 7659 t(Z;1) rearrangement, and other
rearrangements that include the Z chromosome, to confirm the localization of the ev
21 locus. This locus was reported to be localized at Zp24 (Lakshmanan et al. 1992),
while the current linkage maps report it to be more proximal to the centromere
region of the Z chromosome (Bitgood and Somes 1990). This approach will initiate
the process of integration of classical, molecular and physical maps.
REFERENCES.
Bitgood J.J. and R.N.Shoffner RN, Cytology and cytogenetics.
and genetics, 1st ed (Crawford RD, ed). New
York:
1990.
In: Poultry breeding
Elsevier;
401-427.
Bitgood J.J. and R.G. Somes. Linkage relationships and gene mapping. In: Poultry
breeding and genetics, 1st ed (Crawford
RD, ed). New York: Elsevier; 469495. 1990.
21
/
"'
Bumstead, N. and J. Palyga. A preliminary
Genomics 13:690-697. 1992.
linkage map of the chicken genome.
Crittenden, L.B., L. Provencher, L. Santangelo, I. Levin, H. Abplanalp, R.W. Briles,
W.E. Briles and J.B. Dodgson. Characterization
of a Red Jungle Fowl by
White Leghorn backcross reference population for molecular mapping of the
chicken genome. Poultry Science 72:334-348. 1993.
Fechheimer N.S. Chromosomes of Chickens. In: Domestic animal cytogenetics, 1st
ed (McFeely RA, ed). San Diego, California: Academic Press Inc; 170-207.
1990.
Hutt, F.B. Genetics of the fowl. VI. A tentative chromosome map. Neue Forsch.
Tierzucht Abstam. (Duerst Festschrift) pp 105-112. 1936.
Kao, F. T. Identification of chicken chromosomes in cell hybrids formed between
chick erythrocytes and adenine-requiring
mutants of Chinese hamster cells.
Proc. Natl. Acad. Sci. USA 70:2893-2898. 1973.
Lakshmanan,
N., F.A. Ponce de Leon, J.R. Smyth and E.J. Smith."Chromosomal
assignment of the ev21 locus in chicken using fluorescent in situ suppression
hybridization
(FISH)".
Proceedings.
10th European
Colloquium
on
Cytogenetics of Domestic Animals. Utrecht University. The Netherlands. p.
10, 1992.
Levin, I., L. Santangelo, H. Cheng, L.B. Crittenden and J.B. Dodgson. An Autosomal
Genetic Linkage Map of the Chicken. J. of Heredity 85:79-85. 1994.
Lichter,
P., T. Cremer, J. Borden, L. Manuelidis and D.C. Ward. Delineation of
individual human chromosomes in metaphase and interphase cells by in situ
suppression hybridization using recombinant DNA libraries. Hum. Genet.
80:224-234. 1988.
Lichter, P., C.C. Tang, K. Call, G. Hermanson, G.A. Evans, D. Housman and D.C.
Ward.
High-resolution
mapping of human chromosome
11 by in situ
hybridization with cosmid clones. Science 247:64-69. 1990.
Ponce de Leon, F.A., Y. Li and E.J. Smith. Reassignment of the evl locus by high
resolution chromosomal in situ localization. Poultry Science 70(1):95, 1991.
Ponce de Leon FA, Li Y, Weng Z. Early and late replicative chromosomal
patterns of Gallus domesticus. J. of Heredity 82:36-42. 1992.
banding
Ponce de Leon, F.A. and J.M. Robl. Microisolation and Microcloning Of the Bovine
X-chromosome. 23rd International Conference on Animal Genetics. ISAG
Interlaken, Switzerland, University of Berne, p 62, 1992.
Saunders, R.D.C., D.M. Glover, M. Ashburner, I. Siden-Kiamos, C. Louis, M.
Monastirioti,
C. Savakis and F. Kafatos. PCR amplification
of DNA
microdissected
from a single chromosome
band: a comparison
with
conventional microcloning. Nucleic Acids Research 17:9027-9037. 1989.
Zartman, D.L. Location of the pea comb gene. Poultry Science 52:1455-1462.
22
23
FIG. 2
Strategy for Screening and
Identification of Chicken
Chromosome 1 Cosmid Clones
®
End Labeling '_
®
®
®
®
®
®
Chromosome-cocktail
Probe
"
®
-.
1
T
®
®
®
®
®
®
Denaturation
®
®
Cot 1 DNA
®
Reanneling
Cosmid Library
Screening
24
-FIG. 3
FISH Assignments of
Chromosome 1 :osmid Clones
UMA0071
I
56
UMA0014
UMA0037
2
4
2.
_0004
I
UMA0028
3
5
_0045
UMA0010
UMA0009
UMA0074
UMA0032
UMA0030
UMA0036
UMA0027
UMA0024
/_
12
34
.s
UMA0082
UMA0008
UMA0073
2
UMA0021
UMA0020
4
[
25
1
1
2
3
4
I
Question:
Chris Tuggle, ISU
Have you used a newly exported technique called PCR-in situ (Troyer, et al, 1994)?
Response: A. Ponce de Leon
No, DISC-PCR as the technique has been named, has been recently developed. Even
though it allows assignment of small fragments of DNA by PCR amplification directly
from chromosomes, it also requires the observation of a very large number of metaphase
plates and statistical analysis of signals observed to determine exact localization.
The technique does not allow direct identification of chromosomes because, as of now,
chromosomal banding pattern can not simultaneously be visualized. However, it is a
promising technique and other improvements might be coming later.
Question:
J. Zhu
1)
Did labeling more than one nucleotide increase sensitivity of staining?
2)
How many bases should be apart to be able to be recognized by antibodies?
Response:
A. Ponce de Leon
1)
Using more than one biofinalated nucleotide increased the sensitivity of the FITC
signal. Best results were obtained with the use of Biotin-16-dNTP and Biotin-14dATP.
2)
The system we use requires AVIDIN-FITC and there is no stereochemical
difficulty with regard to distance between biofinalated nucleotides. The 16 and 14
carbon chain to which Biotin is attached reduces steriochemical interference in a
very significant way.
26
Comment: L. Crittenden
Seven Chromosome cosmid clones were sent to East Lansing by Dr. Ponce de Leon.
These were used as probes for molecular genetic mapping. One was a single locus probe
and allowed us to assign a linkage group to the long arm of chromosome 1 for the first
time, thus this approach can be used to assign linkage groups from the molecular genetic
map to chromosomes.
Speaker: A. Ponce de Leon
Question: David Vaske
With the microscoping/microisolation techniques, how many of the chicken chromosomes
are large enough to be isolated (and identified) for production of the chromosome specific libraries?
Response: A. Ponce de Leon
Eight pairs ofautosomes and the sex chromosomes will be in time, microisolated for the
generation of chromosome - specific library. The remaining chromosome are called
micro-chromosomes and contain 35% of the chicken genome. The best approach would
be to micro-isolate all micro-chromosomes together to make a micro-chromosome specific
DNA library.
27
Question: E. Smith
For your primary library wouldn't a PI or BAC library (probably not a YAC for now) bc
better? and cosmids only for say, sub cloning?
Response: A. Ponce de Leon
Yes, it would be, however costs to produce a PI or a BAC library are also greater than for
a cosmid library. In an environment where research dollars are tight having a cosmid
library is good enough.
28