Gene Isolation and Mapping Protocols

314 Pages·1996·20.407 MB·English

by  Jacqueline Boultwood

1
Gene Mapping Goes from FISH to Surfing the Net
John Valdes and Danilo A. Tagle
1. Introduction
The amval of the second millenmum will usher m unsurpassed information
and knowledge of our genetic constitution, and will promise to revolutronize
basic research and molecular medicine. The road toward a complete under-
standing of our genetic makeup is largely the fruit of the Human Genome
Project that has mmated, advanced, and made major strtdes in constructing
genetic and physical maps of humans and other model organisms. Already
enttre genomic sequences of a few prokaryottc organisms have become avarl-
able with efforts toward completion of the budding yeast not too far behind.
Gene mapping and identification are critical steps m this ambitrous undertak-
ing. Unfortunately, the identrficatton of genes, especially those responsible for
the vast majority of inherited human disorders, must often proceed without any
knowledge of then biochemical functions. To wit, positional clomng (I) has
taken center stage toward the initial steps m the molecular characterization of
the estimated 100,000 genes in the human genome. This approach has gar-
nered over 60 “diseased” genes thus far, with many more to come as the pro-
cess becomes more streamlined. Despite having achieved in the last several
years a framework of genetic and physical maps of the human genome, none-
theless the efficient and comprehensive isolation of transcribed sequences
within large targeted genomic intervals remains a formidable task. The numer-
ous chapters in this book document the creativity and ingenuity of various
investigators and laboratories m this global effort. Our aim in this introductory
chapter is to give an overvrew of gene mapping and assessw here approaches
in gene isolation are headed m the near future.
From Methods IR Molecular B/o/ogy, Vol 68 Gene lsolatfon and Mapprng Protocols
Edlted by J Boultwood Humana Press Inc , Totowa, NJ
1
Valdes and Tag/e
7.7. ldenfifying and Defining the Chromosome of Interest
The mapping of a gene that contains disease-causing mutations frequently
begins with the assignment of the gene to a single chromosome or to a specific
subchromosomal region. Chromosomal gene assignments can be accomplished
in several ways. For diseases where a large collection of affected families
exists, the gene can be locahzed by lmkage analysts which involves studying
the segregation pattern of the disease phenotype with selected genetic markers
within a pedigree. Statistical methods are used to determine the likelihood that
the marker and disease are segregating Independently. If the chance of mde-
pendent segregation 1sC l in 1000 (an LOD score of 3), then lmkage 1sa ssumed.
Identification of recombinant families using addmonal polymorphrc markers
allows further delineation of the lmked interval. Linkage analysis has shown
widespread success m mapping monogenic disorders that show clear Mende-
lian inheritance patterns. The same principles are now bemg applied to poly-
gemc diseases (those that show complex genetic patterns, likely owing to
multiple genes and/or environmental factors acting m combmation), but this
has proven difficult in practice (2). Proposed soluttons have mcluded use of
standardized ascertainment and the incorporation of interference models (3,4),
inclusion of larger sample sizes, or use of genetically homogeneous popula-
tions in lmkage disequilibrium studies (5).
Human-rodent somatic cell hybrids (either monochromosomal, regional/dele-
tion, or radiation-reduced mapping panels) provide a convenient resource for
mapping of genes by hybridization or polymerase chain reaction (PCR). Hybrid
cell lines have also been useful in genetic complementation studies, such as in
xeroderma plgmentosa and m Niemann-Pick disease (6). Aside from mapping,
radiation hybrids provide additional information about the order and distance
of markers/genes (7,s) where segments of DNA that are farther apart on a chro-
mosome are more likely to be broken apart by radiation and thus segregate
independently in the radiation hybrid cells than rf they were closely linked
together. Fluorescence in situ hybridization (FISH) is also widely used to
determine the chromosomal map location and the relative order of genes and
DNA sequences within a chromosomal band. Unlike hybrid panel mapping
where a cDNA clone or PCR primers are all that is needed, larger genomic
clones, such as cosmids, are needed when mapping via FISH. However FISH
can readily provide more precise regional mapping than regional or radiation
panels. FISH can also detect aneuploidy, gene amplification, and subtle chromo-
somal rearrangements. Discovery of a patient whose inherited disease has
resulted from a visible chromosomal abnormality has often been the ‘Jackpot”
that has accelerated efforts to clone the causal gene (9, IO). The ability to map
by FISH most chromosomal translocations that interrupts or inactivates the
Gene Mapping Goes from FISH to Surfing the Net 3
gene has tremendous utility m the field of cancer genetics (II), where molecu-
lar events leading to the loss of tumor suppressor genes (12) or the generation
of fusion genes (13) can often be detected at the chromosome level. Usmg
FISH on normal metaphase spreads, comparative genomic hybridization
(CGH) allows total genome assessment of changes m relative copy number
(regions of chromosomal loss, gain, or amplificattons) of DNA sequences using
DNA probes derived from tumor cells (14). CGH has the potential to identify
previously unknown regions involved m tumorigenesis.
1.2. Defining and Cloning the Physical Region
Once a genomic interval has been defined for a disease locus, the gene map-
ping efforts now shift toward constructmg a physical map of the candidate
region, determining accurate distances between markers, and cloning the
genomic segment m large insert clones. Physical distances can then be estab-
lished and correlated with the genetic distance (e.g., if two marker probes
hybridize to the same 250-kb fragment, then their maximum dtstance apart
must be 250 kb). Physical distances between genomic markers can be refined
with pulsed-field gel electrophoresis (PFGE) and a combination of rare cutting
restriction enzymes. Because such enzymes occur in GC-rich sequences, the
location of CpG islands, which are likely landmarks for expressed genes, can
then be determined. The pulsed-field maps also provide a reliable method for
verifying the extent of coverage of overlapping clones within a contig in rela-
tion to the actual genomic distance. PFGE can also be used to compare patient
and normal DNA samples, looking for genomic abnormalities that may have
been too small to be detected by cytogenetic techniques (13).
In long-range physical mapping, yeast artificial chromosomes (YACs) are
the cloning library of choice because of their larger insert size, which means
that fewer markers and clones are required to anchor and assemble the contig
(15,26). Where a dense ordered array of markers is available, bacterial artifi-
cial chromosomes (BACs), Pls, or even cosmids are preferred for screening
despite their smaller insert sizes( 120 kb for BACs, 95 kb for P 1s and 40 kb for
cosmids) because of their ease in purifying DNA, relative stability, and low
frequency of chimerism compared to YAC clones. Genomic clones isolated
for the candidate interval are analyzed for insert size and for degree of overlap
by marker content mapping using sequence-tagged sites (STSs) and repetitive
element fingerprint patterns. The clones or derivatives of it can be used as
probes for chromosome walking until full coverage of the candidate interval
are obtained. More importantly, these genomic clones provide a readily avail-
able source of DNA for isolating additional markers, for use as FISH or
hybridization probes, for generating sequence data, and for gene identification.
4 Valdes and Tag/e
1.3. Gene lsola tion
Genetic linkage analysis and physical mapping experiments can often
resolve the rough locatron of a gene to a region of 0.5-l centrmorgan (eqmva-
lent to a frequency of 1 recombinant/l00 meloses), which IS approx 1 Mb.
Such an interval may contain from 3G.50 genes, and rdenttfymg all the genes
n-r such a region and finding the causative gene for the disorder has been a
major bottleneck m most posmonal cloning projects. The choice of which gene
cloning strategies to utrhze often depends on the available resources in a given
laboratory. The common gene hunting methods can be divided mto hybrtdrza-
non-based and functional detection of sequences involved m RNA splicing.
Exon trapping identifies putative transcribed sequencesf rom genomtc clones
(often cosmlds as starting templates) based on splrcmg signals present m
exon-mtron junctions. No assumptrons are made regarding the tissue-specific
pattern of expression of a given gene or of its level of expression. The targeted
exons can be internal (17,18) or directed toward the 3’-termmal exon (19).
Numerous labs have applied the method successfully for both gene lsolatton
(20,21) and mapping intron-exon boundaries of known genes (22).
Transcribed sequencesm genomtc DNA can also be detected by either using
labeled cDNAs as hybridization probes on arrayed genomic clones (23) or the
converse, where genomtc clones are used as probes against cDNA libraries
(24,25). The former approach has taken on numerous permutations where the
genomic YAC clones are either immobrlized on filters (26,27), brotmylated
(28-301, or used in solutron hybrrdizatron schemes (32-34). These methodolo-
gies assume some prior knowledge of the targeted gene’s expression level,
since moderately to abundantly expressed messagesa re those usually obtained,
as well as an idea on the proper tissue source of library to screen. Because the
techniques are hybrrdrzatron-based, problems with sticky or GC-rich cDNAs,
repeat sequences, and pseudogenes and related family gene family members
frequently accompany the final product.
None of the aforementtoned methodologtes are expected to garner full-
length clones. The end points using these techniques are for the most part small
exons or cDNA fragments that can then serve as additional expressed sequence
tagged sites (ESTs) or probes for rsolatmg larger clones
Other gene cloning strategies take advantage of certain features m the
genomlc DNA or transcript. One such feature would be CpG islands that are
areas of the human genome where the CpG dinucleotide is enriched (1 O-20
times greater than other regions). CpG islands tend to be associated with
the 5’-ends of genes and can therefore provide a means of tsolatmg those genes.
A recent survey of 375 genes m the GenBank database demonstrated that
almost all housekeeping genes, and about 40% of tissue-specific genes are
Gene Mapping Goes from FISH to Surfing the Net 5
associated wtth these Islands (3.5). These Islands can be isolated by rare-cutting
enzymes (36-38) or by PCR (39), and used as hybrrdrzatron probes against
cDNA libraries. Another feature would be the differential expression pattern
of genes in certain tissues. Subtraction techmques (40,41) have been used to
isolate genes spectfic to one particular tissue source or developmental stage,
This technique involves the use of a target cDNA hbrary (derived from a tissue
where the desired gene IS likely to be expressed) and a drover cDNA library to
subtract out most ubiquitously expressed sequences.D ifferential display (42-44)
is another method for isolating genes that are unique to a partrcular cell
type or developmental stage and allows the analysts of expressron patterns of
multiple cell types.
A third feature takes advantage of mutants m model organisms whose
phenotype resembles that in human. The mouse genome (as well as that of
other organisms) is also being investigated as part of the Human Genome
Project. Mouse genetic studies are able to take advantage of selective breeding,
short generation times, and backcrosses (matmg between two mice, one of
which is homozygous for a recessive tract, in order to establish the genotype of
the first). One possible approach to mappmg a gene is to isolate the mouse
homolog, determine its genetic localization within the mouse genome, and then
focus efforts on the part of the human genome to which it corresponds. Com-
parative mapping between the mouse and human is fairly well defined: The
entire genome can be separated into 68 homologous chromosomal regions (4.5,).
The observatron and characterizatron of naturally occurrmg mouse mutants
have also supplied model systems (46), as well as acceleratmg the search for
human disease genes (45).
1.4. Future Directions
There is no doubt that the number of genes being cloned by positional clon-
ing approaches is increasing at a rapid rate (5). Most of these genes have been
obtained using the methodologies outlined in this chapter. However newer
resources being made accessible through the Human Genome Project are
promising even to accelerate gene mapping and isolation at a more rapid rate.
With the increasing resolutton of the chromosome physical maps, it is now
feasible to embark on large-scale genomic sequencing (47). This has become
possible despite the lack of significant improvement in sequencing methodol-
ogy, but through a combination of faster computational machines to store and
analyze the data, ready availability of sequence-ready cosmtd clones and their
derivatives, and dense mapping information to help minimize overlap of
cosmid templates. Large-scale sequencing of genomtc clones has been com-
;sleted for a number of prokaryotic organisms (48,49) and implemented for
6 Valdes and Tag/e
diseased loci (50) as an additional gene searching tool. Sequences are queried
to the sequence databases and fed to the Gene Recognition and Analysis
Internet Link (GRAIL) server for exon prediction through computational analy-
sis of the sequence (51,.52).
Another critical development is the concerted effort to develop a transcript
map of the human genome that involves sequencing of human cDNA clones by
the Washington University Genome Sequencing Center under the auspices of
Merck (Whitehouse, NJ) (53). The centerpieces of this undertaking are the
oligo(dT)-primed, directionally cloned and normalized cDNA clones from vari-
ous tissue sources (54,55). Concomitant with the sequencing are efforts to
develop these sequences into gene-based STSs, and place them on the physical
map via YACs (56,57) and radiation hybrid maps. Although attempted in the
past on a limited scale, it is projected that this endeavor will generate approx
400,000 ESTs by early this year (53). The sequences, mapping information,
and homology results are easily accessible via World Wide Servers in the
Internet. As the number of the mapped cDNAs increase, these ESTs automati-
cally become candidate genes if they so happen to fall in an interval linked to a
disease locus. The tremendous potential of this resource can be gleamed from
recent statistics obtained by National Center of Biotechnology Information at
the National Institutes of Health that 79% of positionally cloned genes are
actually represented in the EST database (dBEST at http://www.ncbi.
nlm.nih.gov/dbEST/index.html). Positional cloning will soon be simplified to
a positional candidate approach where linkage of a particular monogenic or
polygenic disorder to a particular chromosomal subregion will be followed by
a survey of the interval for any interesting ESTs (5).
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