The gene orders on human chromosome 15 and chicken chromosome 10 reveal multiple inter- and intrachromosomal rearrangements.

Comparative mapping between the human and chicken genomes has revealed a striking conservation of synteny between the genomes of these two species, but the results have been based on low-resolution comparative maps. To address this conserved synteny in much more detail, a high-resolution human-chicken comparative map was constructed from human chromosome 15. Mapping, sequencing, and ordering of specific chicken bacterial artificial chromosomes has improved the comparative map of chromosome 15 (Hsa15) and the homologous regions in chicken with almost 100 new genes and/or expressed sequence tags. A comparison of Hsa15 with chicken identified seven conserved chromosomal segments between the two species. In chicken, these were on chromosome 1 (Gga1; two segments), Gga5 (two segments), and Gga10 (three segments). Although four conserved segments were also observed between Hsa15 and mouse, only one of the underlying rearrangement breakpoints was located at the same position as in chicken, indicating that the rearrangements generating the other three breakpoints occurred after the divergence of the rodent and the primate lineages. A high-resolution comparison of Gga10 with Hsa15 identified 19 conserved blocks, indicating the presence of at least 16 intrachromosomal rearrangement breakpoints in the bird lineage after the separation of birds and mammals. These results improve our knowledge of the evolution and dynamics of the vertebrate genomes and will aid in the clarification of the mechanisms that underlie the differentiation between the vertebrate species.

Two-dimensional screening of the Wageningen chicken BAC library.

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

Extending the chicken-human comparative map by placing 15 genes on the chicken linkage map.

To increase the number of type I loci on the chicken linkage map, chicken genes containing microsatellite sequences (TAn, CAn, GAn, An) were selected from the nucleotide sequence database and primers were developed to amplify the repeats. Initially, 40 different microsatellites located within genes were tested on a panel of animals from diverse breeds, and identified 17 polymorphic microsatellites. These polymorphisms allowed us to add 15 new genes to the chicken linkage map. In addition, two genes were added to the chicken map by fluorescent in situ hybridization. As the map position of the human homologues of 13 of these genes is known, these markers extend the comparative map between chicken and man. Our results confirm and refine conserved regions between chicken and man on chicken chromosomes 2 and 7 and on linkage group E29C09W09. Furthermore, an additional conserved region is identified on chromosome 7.

Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken.

A chicken embryonic cDNA library was screened with a (TG)13 probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)13 positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.

New microsatellite markers in chicken optimized for automated fluorescent genotyping.

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4.0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).

Development and mapping of polymorphic microsatellite markers derived from a chicken brain cDNA library.

Until now the genetic linkage map in chicken has ben based mainly on random genomic markers. The addition of expressed sequence tags (ESTs) to the genetic linkage maps is becoming more important because ESTs can form the basis for comparative mapping studies. This may be helpful for the detection of candidate genes for quantitative trait loci (QTLs). In our study we used a (TG)13 repeat as probe for the detection of microsatellites in a chicken brain cDNA library. After hybridization 0.15% of the cDNA clones gave a positive signal. The cDNA complexity of the library was high; of the 90 cDNA clones that were sequenced 60 occurred only once. For 29 clones primer sets for the polymerase chain reaction could be developed. Twenty-one microsatellites were polymorphic on one or more of the test panels and 15 markers could be mapped on either or both of the international reference families. Because sequence homology between chicken and mammalian cDNAs is sometimes low it was difficult to assess the level of sequence homology that indicated a true homologous transcript. In our study seven cDNA cones, of which three could be mapped, showed a relatively high percentage of sequence homology with sequences found in other species. Because sequencing and mapping of expressed sequence tags in human and mouse is progressing very rapidly, it is predicted that further information will soon be readily available. Therefore, increasing the number of expressed sequences on the chicken genetic linkage map will be of value for comparative mapping studies in the near future.

Characterization of a GlyCAM1-like gene (glycosylation-dependent cell adhesion molecule 1) which is highly and specifically expressed in the lactating bovine mammary gland.

A bovine cDNA library, derived from the mammary gland of a lactating cow, was screened for identifying transcripts that specifically occur during lactation by means of differential hybridisation. Several of the clones isolated by this procedure shared 55 and 57% similarity with the mouse and rat glycosylation-dependent cell adhesion molecule 1 (GlyCAM1) cDNAs, respectively. Although the mouse and cattle proteins showed an overall similarity of only 41%, two specific regions of the proteins showed 83 and 81% similarity, respectively. The bovine protein also showed 55% similarity with a small protein isolated from the whey fraction of camel milk. Northern blot analysis showed that high-level expression of this gene was only observed in the mammary gland of lactating cows. The complete gene was isolated from a bovine genomic library and its organisation was determined. The gene was 2.5 kb in length and split into four exons. The size and organization of the gene as well as the position of the introns was identical to that of the mouse GlyCAM1 gene. In accordance with the tissue-specific expression of this gene in the mammary gland of lactating animals, potential mammary gland factor (MGF) binding sites were present in the promoter region of the gene. Based on the data presented in this study it is highly likely that this gene is the bovine homologue to the rat and mouse GlyCAM1 genes.

The complete sequence of the gene encoding bovine alpha s2-casein.

From a bovine genomic library, five overlapping clones, spanning some 50 kb, have been isolated. These clones contain the complete alpha s2-casein-encoding gene (alpha s2ca) and its 5' and 3' flanking regions. The nucleotide (nt) sequence of the complete gene including 2510 bp of the 5' flanking region and 276 bp of the 3' region has been determined. The total length of alpha s2ca appears to be 18483 bp and, therefore, it is the longest of the four bovine casein-encoding genes. The alpha s2ca gene is comprised of 18 exons ranging in size from 21 to 266 nt. There are 16 Alu-like artiodactyla retroposons inserted at ten different locations within the gene. About 14% of the gene is composed of these repetitive sequences. Although the organization of alpha s2ca appears to be similar to that of the alpha s1-casein-encoding gene (alpha s1ca), sequence comparisons and the length of the exons indicate that it is more closely related to the beta-casein-encoding gene. Furthermore, it is shown that both genes could have evolved from a common ancestor by means of internal duplications.

Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle.

The nucleotide sequence of the bovine MHC class II alpha genes: DRA, DOA, and DYA.

The nucleotide sequence of the exons 2, 3, and 4, and parts of the intervening sequences of a BoLA-DRA and -DQA gene and one other class II BoLA-A gene have been determined. The structure of the BoLA-DRA and -DQA gene was found to be very similar to that of the corresponding human HLA class II genes. An analysis of the structure of the other class II BoLA-A gene showed that this A gene was clearly very different from both the human A genes and the bovine DRA and DQA genes. The results indicate that this other type of class II A gene probably represents the class II gene that has already been identified in restriction fragment length polymorphism (RFLP) studies as BoLA-DYA. Since no clear homologue of this presumed BoLA-DYA gene was found among the human HLA class II genes, these results indicate that, at least as far as the A genes are concerned, a distinct class II gene is present in cattle.

The nucleotide sequence of bovine MHC class II DQB and DRB genes.

The nucleotide sequences of most of the exons and parts of the introns of two BoLA-DQB genes and two BoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The two DQB genes probably represent true alleles. Based on the exons sequenced, both DQB genes and one of the DRB genes seem to be functional. The other DRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occurred in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease associations in cattle.

Cloning of the bovine major histocompatibility complex class II genes.

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.