Sequence scanning chicken cosmids: a methodology for genome screening
Introduction
Recent international efforts to create a linkage map of the chicken genome have been focused towards the identification of traits of agricultural and biological interest. Invariably, by their nature, these traits, such as meat and egg production, are polygenic (quantitative trait loci) and require a high marker density. There are currently over 600 loci mapped on the linkage map, distributed over 2600–3000 cM (Cheng, 1996). With a rapidly expanding number of markers being mapped, studies are increasingly moving towards the production of a molecular map and detailed studies of gene structure and synteny (Burt et al., 1995).
As a result, the chicken genome is proving to be of increasing interest to comparative genomics. It has a genome size of 1.2×109 bp (Tiersch and Wachtel, 1991), one third of that of mammals, of which only 17% consists of repeat sequences. This small size is also, in part, due to a reduction in intron sizes (Hughes and Hughes, 1995). Chicken therefore represents a relatively compact and intermediate evolutionary model placed between mammals and more distant vertebrates under study, such as the puffer fish and Amphioxus.
Chicken has a karyotype consisting of 39 pairs of chromosomes (including the ZZ/ZW sex chromosome pair), which are delimited into three groups, determined on the basis of size : six pairs of large, morphologically distinguishable macrochromosomes (Ma), four pairs of intermediate size, intermediate microchromosomes (I.mi) and 29 pairs of very small individually indistinguishable microchromosomes (mi) (Bloom et al., 1993). Initial impressions that the small microchromosomes were probably the more minor genetic components are currently being revised. Whilst they only constitute 25% of the total DNA, they show an increased concentration of CpG islands (McQueen et al., 1996), a feature associated with mammalian coding regions, are very GC rich, have fewer repeats and are therefore potentially more gene dense than the macrochromosomes. So far, the majority of genes sequenced and localised in chicken have been mapped to the larger chromosomes, particularly chromosome 1 (Dominguez-Steglich et al., 1992a, Klein et al., 1996, Palmer and Jones, 1986, Shaw et al., 1991, Tereba et al., 1991). This is due mainly to technical reasons, but has influenced the perception of gene distribution in chicken.
In order to redress this problem, and gain a rapid insight into the molecular content of the different chromosome types, an intermediate method between the levels of genetic mapping and high-resolution sequencing has been used. Sequence scanning is an established technique for sampling insert DNA content in clones with large insert sizes. However, its effectiveness depends upon the gene density of the organism under study and, in particular, it has proved of great utility in analysing more compact genomes such as the Japanese puffer fish (Fugu rubripes) (http://Fugu.hgmp.mrc.ac.uk/). This report demonstrates the utility of the sequence scanning technique to analyse the chicken genome.
Section snippets
Shotgun cloning
The cosmid DNAs [selected at random by McQueen et al. (1998) from a Clontech male Leghorn library] were isolated using the standard alkaline lysis method (Birmboim and Doly, 1979). One microgram of DNA was sonicated to an approximate size of 500 bp, end-filled with T4 DNA polymerase, PEG precipitated and ligated into a phosphatased blunt-ended vector, either SmaI-cut M13 (Amersham) or EcoRV-cut pBluescript (Stratagene).
Sequence generation: M13 clones
After transformation, M13 clone DNAs were isolated using the Qiagen (BioRobot
Gene mapping
Nineteen cosmids were chosen randomly from a chicken genomic library (McQueen et al., 1998). They were sequence scanned with an average of 48 shotgun clones sequenced per cosmid. The average cosmid insert size was relatively small at 32 kb, hence the sequence data generated approximates to 70% single pass coverage of each cosmid. Table 1 gives the general statistics for the sequence scanning of the 19 cosmids.
All clones were searched against the SWISS-PROT, TREMBL and EMBL databases using BLAST
Discussion
The chicken genome is of great interest in comparative genomics. It represents a relatively compact and intermediate evolutionary model, ideally placed between mammals and the more distant vertebrates under study, such as the puffer fish and Amphioxus. Additionally, it appears to have a complex genomic structure, with two or perhaps three distinct chromosomal sub-types, the macrochromosomes, intermediate microchromosomes and microchromosomes. However, current data on the chicken genome are
Acknowledgements
This work was supported by an MRC Project Grant.
References (35)
- et al.
Genmark: parallel gene recognition for both DNA strands
Comput. Chem.
(1993) - et al.
Chicken genome mapping: a new era in avian genetics
Trends. Genet.
(1995) - et al.
In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene
Genomics
(1992) - et al.
Mapping the βNGF gene in situ to a microchromosome in chicken
Genomics
(1992) - et al.
The dystrophin gene is autosomally located on a microchromosome in chicken
Genomics
(1990) - et al.
CENSOR — A program for identification and elimination of repetitive elements from DNA sequences
Comput. Chem.
(1996) - et al.
Genomic organisation and expression during embryogenesis of the chicken CR1 repeat
Genomics
(1991) - et al.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucleic Acids Res.
(1997) - et al.
The SWISS-PROT protein sequence data bank and its supplement TREMBL
Nucleic Acids Res.
(1997) - et al.
A rapid alkaline extraction procedure for screening recombinant DNA
Nucleic Acids Res.
(1979)
Constant and variable features of avian chromosomes
Experiment files and their application during large-scale sequencing projects
DNA Sequence
Chicken repeat 1 elements contain a pol-like open reading frame and belong to the non-long terminal repeat class of retrotransposons
Proc. Natl. Acad. Sci. USA
Mapping the chicken genome
Poultry Sci.
Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes
Cytogenet. Cell. Genet.
Mapping of the chicken N-CAM gene and a myosin heavy chain gene: avian microchromosomes are not genetically inert reserves of DNA
J. Exp. Zool
Quality not quantity : the pufferfish genome
Hum. Mol. Genet.
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