Α de novo 3.8-Mb inversion affecting the EDA and XIST genes in a heterozygous female calf with generalized hypohidrotic ectodermal dysplasia.

BACKGROUND: In mammals, hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that is characterized by sparse hair, tooth abnormalities, and defects in cutaneous glands. Only four genes, EDA, EDAR, EDARADD and WNT10A account for more than 90% of HED cases, and EDA, on chromosome X, is involved in 50% of the cases. In this study, we explored an isolated case of a female Holstein calf with symptoms similar to HED. RESULTS: Clinical examination confirmed the diagnosis. The affected female showed homogeneous hypotrichosis and oligodontia as previously observed in bovine EDAR homozygous and EDA hemizygous mutants. Under light microscopy, the hair follicles were thinner and located higher in the dermis of the frontal skin in the affected animal than in the control. Moreover, the affected animal showed a five-fold increase in the number of hair follicles and a four-fold decrease in the diameter of the pilary canals. Pedigree analysis revealed that the coefficient of inbreeding of the affected calf (4.58%) was not higher than the average population inbreeding coefficient (4.59%). This animal had ten ancestors in its paternal and maternal lineages. By estimating the number of affected cases that would be expected if any of these common ancestors carried a recessive mutation, we concluded that, if they existed, other cases of HED should have been reported in France, which is not the case. Therefore, we assumed that the causal mutation was dominant and de novo. By analyzing whole-genome sequencing data, we identified a large chromosomal inversion with breakpoints located in the first introns of the EDA and XIST genes. Genotyping by PCR-electrophoresis the case and its parents allowed us to demonstrate the de novo origin of this inversion. Finally, using various sources of information we present a body of evidence that supports the hypothesis that this mutation is responsible for a skewed inactivation of X, and that only the normal X can be inactivated. CONCLUSIONS: In this article, we report a unique case of X-linked HED affected Holstein female calf with an assumed full inactivation of the normal X-chromosome, thus leading to a severe phenotype similar to that of hemizygous males.

Assessing the quality of donor cells: karyotyping methods.

Somatic cell nuclear transfer (SCNT) has a low success rate that rarely exceeds 5 %. Moreover, SCNT requires highly technical skills and may be influenced by the biological material used (oocyte and donor cell quality). Hence, it is crucial to check the normality of the donor cell's karyotype. Numerical and structural chromosome abnormalities are detected by cytogenetic analysis at minimum using G-banding to identify the chromosomes. Here, we describe the classical protocols that are needed to perform complete cytogenetic analyses, i.e., G-banding to identify chromosome aberrations, followed by Fluorescent In Situ Hybridization (FISH) of specific probes for a more sensitive detection and precise identification of the rearrangement.

Accuracy and coverage assessment of Oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (OryCun2.0) and localization of the IGH locus to chromosome 20.

We report on the analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51× whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG, and IGHE sequences in another. Some IGKV, IGLV, and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3-Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa, and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.

A 3.7 Mb deletion encompassing ZEB2 causes a novel polled and multisystemic syndrome in the progeny of a somatic mosaic bull.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

Characterization of the ovine ribosomal protein SA gene and its pseudogenes.

BACKGROUND: The ribosomal protein SA (RPSA), previously named 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR) is a multifunctional protein that plays a role in a number of pathological processes, such as cancer and prion diseases. In all investigated species, RPSA is a member of a multicopy gene family consisting of one full length functional gene and several pseudogenes. Therefore, for studies on RPSA related pathways/pathologies, it is important to characterize the whole family and to address the possible function of the other RPSA family members. The present work aims at deciphering the RPSA family in sheep. RESULTS: In addition to the full length functional ovine RPSA gene, 11 other members of this multicopy gene family, all processed pseudogenes, were identified. Comparison between the RPSA transcript and these pseudogenes shows a large variety in sequence identities ranging from 99% to 74%. Only one of the 11 pseudogenes, i.e. RPSAP7, shares the same open reading frame (ORF) of 295 amino acids with the RPSA gene, differing in only one amino acid. All members of the RPSA family were annotated by comparative mapping and fluorescence in situ hybridization (FISH) localization. Transcription was investigated in the cerebrum, cerebellum, spleen, muscle, lymph node, duodenum and blood, and transcripts were detected for 6 of the 11 pseudogenes in some of these tissues. CONCLUSIONS: In the present work we have characterized the ovine RPSA family. Our results have revealed the existence of 11 ovine RPSA pseudogenes and provide new data on their structure and sequence. Such information will facilitate molecular studies of the functional RPSA gene taking into account the existence of these pseudogenes in the design of experiments. It remains to be investigated if the transcribed members are functional as regulatory non-coding RNA or as functional proteins.

Heat-induced and spontaneous expression of Hsp70.1Luciferase transgene copies localized on Xp22 in female bovine cells.

BACKGROUND: Expression of several copies of the heat-inducible Hsp70.1Luciferase (LUC) transgene inserted at a single X chromosome locus of a bull (Bos taurus) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell. FINDINGS: Transgene copies were mapped to bovine Xp22. In XXLUC female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active XLUC chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic (female) embryos obtained after IVF and in male and female embryos obtained after SCNT. CONCLUSIONS: The XLUC chromosome is normally inactivated but at least part of the inactivated X-linked Hsp70.1Luciferase transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the XLUC chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.

Characterization, chromosomal assignment, and role of LIFR in early embryogenesis and stem cell establishment of rabbits.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine with an important role during early embryonic development, implantation, and as an inhibitor of murine embryonic stem cell differentiation. It exerts its effects by binding to the leukemia inhibitory factor receptor, a heterodimer of two transmembrane proteins, the specific leukemia inhibitory factor receptor subunit, and the common gp130. A partial cDNA clone coding for the membrane-bound form of the specific rabbit leukemia inhibitory factor receptor was isolated from the genital ridge of 13.5 days postcoitum fetus. Fluorescent in situ hybridization analysis revealed that the rabbit leukemia inhibitory factor receptor gene is located on chromosome OCU11p11.1. It has been shown that the membrane-bound rabbit leukemia inhibitory factor receptor mRNA is expressed during embryo implantation but not at earlier developmental stages. Rabbit embryonic stem cell-like line establishment is improved in the presence of LIF, and those cells express both leukemia inhibitory factor and its receptor. The withdrawal of leukemia inhibitory factor results the differentiation of embryonic stem cell-like cells to beating myocardial-like cells. Our findings suggest that the self-renewal mechanism is similar in mouse and rabbit embryonic stem cells, and expands our knowledge on the role of the LIF-LIFR signal pathway in early rabbit embryogenesis and rabbit embryonic stem cell establishment.

Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie.

Scrapie is a transmissible spongiform encephalopathy in sheep and goats. Susceptibility to this neurodegenerative disease is mainly controlled by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to scrapie. On the basis of several studies in Alzheimer and different transmissible spongiform encephalopathy models, HSP90AA1 was chosen as a putative positional and functional candidate gene that might be involved in the polygenic variance mentioned above. In the present work, the ovine HSP90AA1 gene including the promoter and other regulatory regions has been isolated and characterized. Several sequence polymorphisms have also been identified. FISH-mapping localized the HSP90AA1 gene on ovine chromosome OAR19q24dist, which was confirmed by linkage analysis. This chromosome region has been shown to include a quantitative trait loci (QTL) for scrapie incubation period in sheep. Expression analyses were carried out in spleen and cerebellum samples. No differences in the expression of the HSP90AA1 gene were found in any of these tissues (p > 0.05) between control and infected animal samples. Nevertheless, association analyses revealed that several polymorphisms in the 5' and 3' regions of the HSP90AA1 gene were differentially distributed among animals with different responses to scrapie infection. Thus, results presented here support the hypothesis that HSP90AA1 could be a positional and functional candidate gene modulating the response to scrapie in sheep.

Structural and functional analysis of the ovine laminin receptor gene (RPSA): Possible involvement of the LRP/LR protein in scrapie response.

Scrapie is a prion disease affecting sheep and goats. Susceptibility to this neurodegenerative disease shows polygenic variance. The involvement of the laminin receptor (LRP/LR) in the metabolism and propagation of prions has previously been demonstrated. In the present work, the ovine laminin receptor gene (RPSA) was isolated, characterized, and mapped to ovine chromosome OAR19q13. Real-time RT-PCR revealed a significant decrease in RPSA mRNA in cerebellum after scrapie infection. Conversely, no differences were detected in other brain regions such as diencephalon and medulla oblongata. Association analysis showed that a polymorphism reflecting the presence of a RPSA pseudogene was overrepresented in a group of sheep resistant to scrapie infection. No amino acid change in the LRP/LR protein was found in the 126 sheep analyzed. However, interesting amino acid positions (241, 272, and 290), which could participate in the species barrier to scrapie and maybe to other transmissible spongiform encephalopathies, were identified by comparing LRP/LR sequences from various mammals with variable levels of resistance to scrapie.

Characterization of the genomic region containing the Shadow of Prion Protein (SPRN) gene in sheep.

BACKGROUND: TSEs are a group of fatal neurodegenerative diseases occurring in man and animals. They are caused by prions, alternatively folded forms of the endogenous prion protein, encoded by PRNP. Since differences in the sequence of PRNP can not explain all variation in TSE susceptibility, there is growing interest in other genes that might have an influence on this susceptibility. One of these genes is SPRN, a gene coding for a protein showing remarkable similarities with the prion protein. Until now, SPRN has not been described in sheep, a highly relevant species in prion matters. RESULTS: In order to characterize the genomic region containing SPRN in sheep, a BAC mini-contig was built, covering approximately 200,000 bp and containing the genes ECHS1, PAOX, MTG1, SPRN, LOC619207, CYP2E1 and at least partially SYCE1. FISH mapping of the two most exterior BAC clones of the contig positioned this contig on Oari22q24. A fragment of 4,544 bp was also sequenced, covering the entire SPRN gene and 1206 bp of the promoter region. In addition, the transcription profile of SPRN in 21 tissues was determined by RT-PCR, showing high levels in cerebrum and cerebellum, and low levels in testis, lymph node, jejunum, ileum, colon and rectum. CONCLUSION: Annotation of a mini-contig including SPRN suggests conserved linkage between Oari22q24 and Hsap10q26. The ovine SPRN sequence, described for the first time, shows a high level of homology with the bovine, and to a lesser extent with the human SPRN sequence. In addition, transcription profiling in sheep reveals main expression of SPRN in brain tissue, as in rat, cow, man and mouse.

IL-1 family members as candidate genes modulating scrapie susceptibility in sheep: localization, partial characterization, and expression.

Scrapie (SC) is a transmissible spongiform encephalopathy (TSE) in sheep and goats. Susceptibility to this neurodegenerative disease is controlled mainly by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to SC. On the basis of several studies on Alzheimer's disease and different TSE models, and of requirement for correct homeostasis of cytokines in brain, IL1B and IL1RN were chosen as putative positional and functional candidate genes that might be involved in the polygenic variance mentioned above. In the present work, ovine IL1B and IL1RN genes were partially isolated and characterized, including promoter and other regulatory regions. In addition, several sequence polymorphisms were identified. Furthermore, their cytogenetic positions on sheep chromosomes were determined by FISH and confirmed by linkage analysis, localizing both genes in OAR3p22, a region previously described as carrying a QTL for SC incubation period in sheep. Finally, expression analyses were carried out in eight naturally SC-infected and five uninfected sheep with the same genotype for PRNP (ARQ/ARQ). This comparison was performed using real-time RT-PCR in samples of spleen and cerebellum. Results showed differences in the expression of both cytokines in cerebellum (p < 0.05) but not in spleen (p > 0.05).

High-resolution comparative mapping among man, cattle and mouse suggests a role for repeat sequences in mammalian genome evolution.

BACKGROUND: Comparative mapping provides new insights into the evolutionary history of genomes. In particular, recent studies in mammals have suggested a role for segmental duplication in genome evolution. In some species such as Drosophila or maize, transposable elements (TEs) have been shown to be involved in chromosomal rearrangements. In this work, we have explored the presence of interspersed repeats in regions of chromosomal rearrangements, using an updated high-resolution integrated comparative map among cattle, man and mouse. RESULTS: The bovine, human and mouse comparative autosomal map has been constructed using data from bovine genetic and physical maps and from FISH-mapping studies. We confirm most previous results but also reveal some discrepancies. A total of 211 conserved segments have been identified between cattle and man, of which 33 are new segments and 72 correspond to extended, previously known segments. The resulting map covers 91% and 90% of the human and bovine genomes, respectively. Analysis of breakpoint regions revealed a high density of species-specific interspersed repeats in the human and mouse genomes. CONCLUSION: Analysis of the breakpoint regions has revealed specific repeat density patterns, suggesting that TEs may have played a significant role in chromosome evolution and genome plasticity. However, we cannot rule out that repeats and breakpoints accumulate independently in the few same regions where modifications are better tolerated. Likewise, we cannot ascertain whether increased TE density is the cause or the consequence of chromosome rearrangements. Nevertheless, the identification of high density repeat clusters combined with a well-documented repeat phylogeny should highlight probable breakpoints, and permit their precise dating. Combining new statistical models taking the present information into account should help reconstruct ancestral karyotypes.

Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene.

Whey Acidic Protein (WAP) has been identified in the milk of only a few species, including mouse, rat, rabbit, camel, pig, tammar wallaby, brushtail possum, echidna and platypus. Despite intensive studies, it has not yet been found in the milk of Ruminants. We have isolated and characterized genomic WAP clones from ewe, goat and cow, identified their chromosomal localization and examined the expression of the endogenous WAP sequence in the mammary glands of all three species. The WAP sequences were localized on chromosome 4 (4q26) as expected from comparative mapping data. The three ruminant WAP sequences reveal the same deletion of a nucleotide at the end of the first exon when compared with the pig sequence. Due to this frameshift mutation, the putative proteins encoded by these sequences do not harbor the features of a usual WAP protein with two four-disulfide core domains. Moreover, RT-PCR experiments have shown that these sequences are not transcribed and are, thus, pseudogenes. This loss of functionality of the gene in Ruminants raises the question of the biological role of the WAP. Some putative roles previously suggested for WAP are discussed.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.

Construction of a cytogenetically anchored microsatellite map in rabbit.

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.

A comprehensive radiation hybrid map of bovine Chromosome 26 (BTA26): comparative chromosomal organization between HSA10q and BTA26 and BTA28.

In this study, we present a comprehensive 3000-rad radiation hybrid (RH) map of bovine Chromosome (Chr) 26 (BTA26) with 80 markers including 50 genes or ESTs: 44 have an ortholog mapping to human Chr 10 (HSA10) and 29 to mouse Chr (MMU) 7, 10, and 19. Moreover, 12 other HSA10 genes were integrated in a newly developed RH map of BTA28 (seven represent new assignments). The available draft of the mouse genome allowed us to present a detailed picture of the distribution of conserved synteny segments among the three species (human, cattle, and mouse) and to propose a simple model of the comparative chromosomal organization between the long arm of HSA10 and BTA26 and 28. Finally, the INRA bovine BAC library was screened for most of the BTA26 markers considered in this study to provide anchors for the bovine physical map.

Structure and expression of goat GLYCAM1 gene: lactogenic-dependent expression in ruminant mammary gland and interspecies conservation of the proximal promoter.

A macroarray approach used to list genes differentially expressed in goat mammary gland (gestation vs. lactation), other than milk protein genes, allowed us to detect the Glycosylation-dependent Cell Adhesion Molecule 1 (GLYCAM1) gene. GLYCAM1, a member of the glycoprotein mucin family, is a component of the milk fat globule membrane (MFGM). Its complete cDNA and gene sequences were determined and it was mapped by fluorescent in situ hybridization (FISH) on goat and cattle chromosome 5 (CHI5q21 and BTA5q21), and on sheep chromosome 3 (OAR3q21). Northern blot analyses confirmed its differential expression during the development and differentiation of the mammary gland of ruminants with a significantly higher mRNA amount during lactation than during pregnancy. An experimental in vivo induction model for lactation, developed by Kann et al., showed that the expression of GLYCAM1 is hormonally regulated in the mammary gland of ewes. Interspecies comparison of the gene promoter revealed the evolutionary conservation of a short proximal nucleotide sequence encompassing several transcription factor binding sites that could mediate the above-mentioned hormonal regulation.

An extensive and comprehensive radiation hybrid map of bovine Chromosome 15: comparison with human Chromosome 11.

An extensive and comprehensive radiation hybrid map of bovine Chromosome 15 (BTA15) was built with 42 anonymous markers, 3 ESTs, and 49 genes. This work allows us to refine the comparative map between human Chromosome (Chr) 11 (HSA11) and BTA15. Four blocks with a similar gene content and relatively good gene order conservation were identified. The discrepancies are concentrated on closely positioned genes for which discrimination is not possible between mapping resolution limits in either the human or the bovine maps and true local inversions. Using the gene order similarity and the human physical map as starting point, we estimated the overall physical length of BTA15 to be around 75.3 Mb. The INRA bovine BAC library was screened for all the markers ordered on the bovine map, which will provide anchors for future efforts in the construction of a physical map of the bovine genome.Finally, this map contains the majority of publicly available polymorphic markers described for BTA15 and integrates those with comparative mapping information. It should, therefore, constitute a powerful tool in the identification of relevant candidate genes in regions of BTA15 harboring economic trait loci.

Structure of the rabbit alphas1- and beta-casein gene cluster, assignment to chromosome 15 and expression of the alphas1-casein gene in HC11 cells.

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-CSN) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN/BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN/BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN/BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN/BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.