Identification of the ovine KAP11-1 gene (<Emphasis Type="Italic">KRTAP11</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) and genetic variation in its coding sequence

Keratin-associated proteins (KAPs) are a structural component of the wool fibre and form the matrix between the keratin intermediate filaments (KIFs). The gene encoding high sulphur-protein KAP11-1 has been identified in human, cattle and mouse, but not yet in sheep, despite the economic importance of wool. In this study, PCR using primers based on the cattle KAP11-1 gene sequence produced an amplicon of the expected size with sheep DNA. Upon using PCR–Single Stranded Conformational Polymorphism (PCR–SSCP) analysis in 260 sheep, six different PCR–SSCP patterns were detected. Either one or a combination of two banding patterns was observed for each sheep, suggesting they were either homozygous or heterozygous for this gene. Sequencing of the amplicons confirmed the occurrence of six DNA sequences. All of these were unique, and the greatest homology was with KRTAP11-1 sequences from cattle, human and mouse, suggesting that they were derived from the ovine KAP11-1 gene and were allelic variants. The ovine KAP11-1 gene had an open reading frame of 477 nucleotides encoding 159 amino acids. The putative protein was rich in serine, cysteine, and threonine which account for 18.2–18.9, 12.6 and 12.0 mol%, respectively. Of these, approximately 20 of the serine and threonine residues might be phosphorylated. Five nucleotide substitutions were identified, and one was non-synonymous and would result in an amino acid change at a potential phosphorylation site. The genetic variation found in KRTAP11-1 may influence its expression, protein structure, and/or post-translational modifications, and consequently affect wool fibre structure and wool traits.     

Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (<Emphasis Type="Italic">KAP6</Emphasis>) family in sheep

Keratin-associated proteins (KAPs) are structural components of wool and variation in them may affect wool characteristics. In this study, we used PCR-SSCP to analyse the ovine KAP6 family which encodes glycine and tyrosine-rich KAPs. Five unique PCR-SSCP patterns were detected in the 250 sheep investigated. Between two and five patterns were observed in individual sheep and none with only one pattern was detected. This suggests the amplicons were heterogeneous and derived from more than one locus. To analyse these heterogeneous PCR amplicons, a sequencing approach using SSCP to separate individual amplified sequences, was developed. Using this approach, five DNA sequences (A–E) representing five unique PCR-SSCP patterns were obtained. D was identical to a published ovine KAP6-1 sequence (GenBank accession no. M95719), whereas the others were novel, but the closest homology was with KAP6 sequences from human, sheep, goats and cattle. The five ovine KAP6 sequences could be assigned into three distinct groups. B and D were identical to each other, with the exception of a 57-bp deletion/insertion and a single nucleotide polymorphism (SNP) in the 3′-UTR region. These appear to be allelic variants of ovine KAP6-1. A and C could form another group, as they were similar to each other (with only one synonymous SNP), but different to the other sequences. This group appears to be related to a sheep KAP6 amino acid sequence, and represent allelic variation at another KAP6 locus (designated KAP6-2). The remaining sequence E did not show high sequence homology with either the KAP6-1 or KAP6-2 sequences, but exhibited homology with a bovine KAP6-3 sequence, with the exception of a deletion/insertion of 30 nucleotides. This suggests that E represents ovine KAP6-3. This sequence was detected in only 11% of the sheep investigated, suggesting either a KAP6-3 null allele, or failure to amplify allleles. These results suggest that ovine KAP6 is a complex gene family, that is not only comprised multiple loci, but that is also polymorphic.     

Search for variation in the ovine KAP7-1 and KAP8-1 genes using polymerase chain reaction-single-stranded conformational polymorphism screening

Keratins and keratin-associated proteins (KAPs) are large heterogeneous groups of proteins that constitute about 90% of the wool fiber. The genes encoding the high glycine-tyrosine (HGT) KAPs are the first sub-group of KAP genes expressed in the wool follicle and just after expression of the keratin genes. Little is known about variation in these genes, which led us to investigate two HGT-KAP genes, KRTAP7-1 and KRTAP8-1. Polymerase chain reaction-single-stranded conformational polymorphism analysis was used to investigate these genes in 250 Romney-cross sheep. For KRTAP7-1, two unique banding patterns were detected for amplicons that spanned the entire coding region. Sequencing confirmed the presence of two sequences with only one nucleotide difference (c.173G/A) putatively resulting in p.Ser58Asn. One was identical to the published ovine KRTAP7-1 sequence. For KRTAP8-1, five unique banding patterns were detected in an amplicon that spanned the entire coding region. Sequencing revealed five different DNA sequences, all of which were highly homologous to the previously reported ovine KRTAP8-1 sequence. Among these five sequences, four single-nucleotide substitutions were identified and three of them were located in the coding region. One of these was nonsynonymous and would putatively result in p.Tyr34Asn. The variation detected in KRTAP7-1 and KRTAP8-1 may influence their expression or protein structure.     

Ovine keratome: identification,localisation and genomic organisation of keratin and keratin‐associated proteins

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin‐associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST‐derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.     

Characterisation of white and black merino wools: a proteomics study

Wool is an important agricultural commodity with merino wool being rated alongside the finest quality fibres, which include the goat fibres Mohair and Cashmere. Although pigmented wool merinos have become extremely rare, the market for this wool is increasing. In Portugal, there are two merino breeds: white and black, descendants of animals originally bred on the Iberian Peninsula. These breeds have the potential to assist in our understanding of how protein expression relates to wool traits of importance to the textile industry. Herein, we study the characteristics and protein expression profiles of wool from ewes of the Portuguese black and white merino (n=15). Both breeds had very similar results for fibre diameter (25 µm) and curvature (105 to 111°/mm). Significant between-breed differences were found in the two types of keratin-associated proteins (KAPs): high-sulphur proteins (HSPs) and high-glycine–tyrosine proteins (HGTPs). The expression of HSPs, KAP2-3 and KAP2-4, decreased expression in the pigmented animals, whereas KAP13-1 was found in higher amounts. Likewise, the expression of the ultra-high-sulphur proteins, KAP4-3 and KAP4-7-like, was reduced in black sheep to half the levels of the white wools, whereas the HGTPs, KAP6, KAP6-1, KAP6-2 and KAP16-2, were more abundant in black sheep. These results suggest structural differences between the black and white merino wool, because of differences among some KAPs. These differences have important implications for the textile industry.     

Identification of two novel clusters of ultrahigh-sulfur keratin-associated protein genes on human chromosome 11

We analyzed two novel clusters of keratin-associated protein (KAP) genes on human chromosome 11 (11p15.5 and 11q13.5) in which we identified two known human KRTAP5 genes, KerA (=KRN1) and KerB, and nine novel KRTAP5 family genes. RT-PCR analysis of these KAP genes showed preferential expression in human hair root, suggesting these gene products are required for hair formation. Based on the deduced amino acid sequences, all these KAP proteins were classified into an ultrahigh-sulfur (UHS) type KAP with high cysteine content (> 30 mol%). These KAPs also showed high glycine and serine contents (average 24.30 and 21.13 mol%, respectively), distinguishing from other UHS/HS KAP families located on human chromosomes 17 and 21. Dot-matrix analysis revealed a significant similarity between these two KAP gene clusters. We postulated a mechanism by which these two KAP gene clusters are generated via genomic duplication of a primordial gene cluster followed by genetic modification during evolution.     

Haplotypic Diversity Within the Ovine Calpastatin <Emphasis Type="Italic">(CAST)</Emphasis> Gene

Calpastatin (CAST) is a protein inhibitor that acts specifically on calpains and plays a regulatory role in postmortem beef tenderization and muscle proteolysis. Polymorphisms in the bovine CAST gene have been associated with meat tenderness, but little is known about how the ovine CAST gene may affect sheep meat quality traits. In this study, we selected two parts of the ovine CAST gene that have been previously reported to be polymorphic (region 1—part of intron 5 and exon 6, and region 2—part of intron 12), to investigate haplotype diversity across an extended region of the ovine gene. First, we developed a simple and efficient polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method for genotyping region 2, which allowed the detection of a novel allele as well as the three previously reported alleles. Next, we genotyped both regions 1 and 2 of the ovine CAST gene from a large number of sheep to determine the haplotypes present. Nine different haplotypes were found across this extended region of the ovine CAST gene and four haplotypes were identified that suggested historical recombination events within this gene. Haplotypes are typically more informative than single nucleotide polymorphisms (SNPs) for analyzing associations between genes and complex production traits, such as meat tenderness, but the potential for intragenic recombination within the ovine CAST may make finding associations challenging.     

An updated nomenclature for keratin-associated proteins (KAPs)

Most protein in hair and wool is of two broad types: keratin intermediate filament-forming proteins (commonly known as keratins) and keratin-associated proteins (KAPs). Keratin nomenclature was reviewed in 2006, but the KAP nomenclature has not been revised since 1993. Recently there has been an increase in the number of KAP genes (KRTAPs) identified in humans and other species, and increasingly reports of variation in these genes. We therefore propose that an updated naming system is needed to accommodate the complexity of the KAPs. It is proposed that the system is founded in the previous nomenclature, but with the abbreviation sp-KAPm-nL*x for KAP proteins and sp-KRTAPm-n(p/L)*x for KAP genes. In this system "sp" is a unique letter-based code for different species as described by the protein knowledge-based UniProt. "m" is a number identifying the gene or protein family, "n" is a constituent member of that family, "p" signifies a pseudogene if present, "L" if present signifies "like" and refers to a temporary "place-holder" until the family is confirmed and "x" signifies a genetic variant or allele. We support the use of non-italicised text for the proteins and italicised text for the genes. This nomenclature is not that different to the existing system, but it includes species information and also describes genetic variation if identified, and hence is more informative. For example, GenBank sequence JN091630 would historically have been named KRTAP7-1 for the gene and KAP7-1 for the protein, but with the proposed nomenclature would be SHEEP-KRTAP7-1*A and SHEEP-KAP7-1*A for the gene and protein respectively. This nomenclature will facilitate more efficient storage and retrieval of data and define a common language for the KAP proteins and genes from all mammalian species.     

Comparative genomics of the keratin-associated protein (KAP) gene clusters in human,chimpanzee, and baboon

We have previously identified a cluster of 16 genes that encode hair-specific proteins, called keratin-associated proteins (KAPs), located on human Chromosome (Chr) 21q22.3. Here, we have identified similar KAP gene clusters in two primates, chimpanzee and baboon. DNA sequence comparison revealed the common cluster structure consisting of 16 KAP genes for these three primates, but a significant difference was found in the baboon. Baboon possesses a new KAP gene not found in human and chimpanzee, whereas one KAP gene (KRTAP18.12) that exists in human and chimpanzee was lost in baboon, making no change in the total number of KAP genes. Interestingly, the sequence for coding regions are highly variable among species owing to insertions and deletions, resulting in variation of gene size. On the contrary, the sequences for the 5 upstream region are highly conserved among species. These findings suggest that the ancestral KAP gene cluster was composed of 17 genes before the divergence of Old World monkeys (baboon) to the anthropoid (human and chimpanzee). The sequence data described in this paper have been submitted to the DDBJ/EMBL/GenBank data library under accession nos. AP006271–AP006274. (Shinsei Minoshima) Present address: Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan.     

Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98–99%, 94–98%, 92–93% and 82–83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A > T, c.282G > T and c.296C > G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2 + channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P < 0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.     

Identification of SNPs within the sheep <Emphasis Type="Italic">PROP1</Emphasis> gene and their effects on wool traits

Regarding mutations of PROP1 (Prophet of POU1F1) gene significantly associating with combined pituitary hormone deficiency (CPHD) in human patients and animals, PROP1 gene is a novel important candidate gene for detecting genetic variation and growth, reproduction, metabolism traits selection and breeding. The aim of this study was to detect PROP1 gene mutation of the exon 1–3 and its association with wool traits in 345 Chinese Merino sheep. In this study, on the basis of PCR-SSCP and DNA sequencing methods, ten novel SNPs within the sheep PROP1 gene, namely, AY533708: g.45A > G resulting in Glu15Glu, g.1198A > G, g.1341G > C resulting in Arg63Ser, g.1389G > A resulting in Ala79Ala, g.1402C > T resulting in Leu84Leu, g.1424A > G resulting in Asn91Ser, g.1522C > T, g.1556A > T, g.1574T > C, g.2430C > G were reported. In addition, association analysis showed that three genotypes of P4 fragment were significantly associated with fiber diameter in the analyzed population (P = 0.044). These results strongly suggested that polymorphisms of the PROP1 gene could be a useful molecular marker for sheep breeding and genetics through marker-assisted selection (MAS).     

Identification of Allelic Polymorphism in the Ovine Leptin Gene

Leptin is an adipocyte-derived hormone/cytokine that influences the physiological control of numerous biological functions and links nutritional status with both neuroendocrine and immune functions. In livestock, variation in the leptin (LEP) gene has been characterized in cattle and pig, but it has not been reported in sheep. In this study, variation in the exon 3 coding sequence of the ovine LEP gene was investigated by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) analysis and DNA sequencing. Five novel SSCP patterns, representing five different sequences, were identified under a combination of two different electrophoresis conditions. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology with the LEP sequences from a variety of species, suggesting that these sequences represent alleles of the ovine LEP gene. Four single nucleotide polymorphisms (SNPs) were detected, and three of these resulted in amino acid changes. Variation detected here might have an impact on leptin activity and function.     

Allelic variation in ovine fatty acid-binding protein (FABP4) gene

The fatty acid-binding protein 4 (FABP4) plays a role in lipid metabolism and has been implicated in intra-cellular lipid transport. While FABP4 variation has been reported in some species, variation in the coding sequence has not been reported in sheep. In this study two regions of ovine FABP4 were analysed using PCR-SSCP and sequencing. Five different PCR-SSCP patterns, representing five specific sequences (A ₁ –E ₁ ) were detected in region 1 (exon 2–intron 2) with sequence analysis revealing three nucleotide substitutions and one deletion in the intron. In region 2 (exon 3–intron 3), four different PCR-SSCP patterns (A ₂ –D ₂ ) were observed and four nucleotide substitutions were revealed. In total, fourteen haplotypes through both regions were defined. There was a difference (P?<?0.001) in allele frequencies between two selection lines of Coopworth sheep that have been bred over many generations to be lean or fat. In region 1, A ₁ and B ₁ were most common (at a frequency of 50 and 30?% respectively) in the fat line, whereas these two variants were absent or rare in the lean line in which C ₁ predominated (89?%). In region 2, C ₂ was the most common variant (59?%) in the lean line but was absent in the fat line, whereas B ₂ was predominant (83?%) in the fat line but was rare (3?%) in the lean line. These results indicate that ovine FABP4 is polymorphic and suggest further analysis is required to see if the variation detected affects fat deposition in sheep.     

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.     

Key determinants affecting sheep wool biodegradation directed by a keratinase-producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strain

OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.     

Characterization and polymorphism of Keratin Associated Protein 1.4 gene in goats

Keratin-associated proteins (KAPs) are among the main structural components of the animal fibers and form semi-rigid matrix wherein the keratin intermediate filaments (KIFs) are embedded. Variation in the KAP genes has been reported to affect the structure of KAPs and hence fiber characteristics. As no information is available on this gene in Capra hircus therefore, present work was undertaken to characterize and explore the different polymorphic variants of KAP1.4 gene at DNA level in different breeds/genetic groups of goats of Kashmir. Cashmere (Changthangi, 30 animals) and non-Cashmere (Bakerwal and Kargil goats, 20 animals each) goats formed the experimental animals for the study. Single strand conformation polymorphism technique was employed for exploring variability at gene level. On exploring the size variability in KAP1.4 gene between Ovine and Caprine, it was concluded that sheep KAP1.4 gene has a deletion of 30 nucleotides. In comparison to published nucleotide sequences of sheep, goat sequences explored are differing at positions 174, 462 and 568 and at these positions “G”, “T” and “T” nucleotides are present in sheep, but are replaced by “A”, “C” and “C” respectively, in goats. By SSC studies, two genotypes were observed in each genetic group and in Bakerwal goats the genotypes were designated as A1A1 (0.40) and A1A2 (0.60) and were formed by two alleles A1 (0.70) andA2 (0.30). The different SSC patterns observed in Kargil goats were designated as B1B1 (0.35) and B1B2 (0.65) genotypes with frequencies of B1 and B2 alleles as 0.675 and 0.325, respectively. Similarly, two genotypes C1C1 (0.60) and C1C2 (0.40) were observed in Changthangi goats and the frequencies of C1 and C2 alleles were 0.80 and 0.20, respectively. These alleles were later confirmed by sequencing. The sequences of these alleles are available in NCBI under Acc. No's. JN012101.1, JN012102.1, JN000317.1, JN000318.1, JQ436929 and JQ627657. It was concluded that all the alleles observed in a breed were unique to the breed. The designated A1 and A2 alleles of Bakerwal goats differ from each other at positions 245 and the nucleotides observed were “C” or “A” and at position 605 of the nucleotide sequence “T” or “C”, were observed. The designated B1 and B2 alleles of Kargil goats differed from each other at positions 224, 374, 375 and 521. The nucleotides observed in two SSC pattern were C→G, A→G, G→A and T→C, respectively. The designated C1 and C2 alleles of Changthangi goats differed from each other at one position 440 with the change of “A”→“C”.     

A Cluster of Keratin-Associated Proteins on Mouse Chromosome 10 in the Region of Conserved Linkage with Human Chromosome 21

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes,Krtap12-1,has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein.Krtap12-1is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family.Krtap12-1is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, betweenITGB2(integrin β2) andPFKL(the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.