Search

Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder. Through the study of a mouse model, we identified a mutation in the 5'-untranslated region of the hairless (HR) gene in patients with MUHH in a Caucasian family. The corresponding mutation, named 'hairpoor', was found in mutant mice that were generated through N-ethyl-N-nitrosourea mutagenesis. Hairpoor mouse mutants display partial hair loss at an early age and progress to near alopecia, which resembles the MUHH phenotype. This mutation conferred overexpression of HR through translational derepression and, in turn, decreased the expression of Sfrp2, an inhibitor of the Wnt signaling pathway. This study indicates that the gain in function of HR also results in alopecia, as seen with the loss of function of HR, via abnormal upregulation of the Wnt signaling pathway.

The hair follicle develops from the primitive embryonic epidermis as a result of complex epithelial-mesenchymal interactions. The full follicle, consisting of epithelial cylinders under control of a proximal lying mesenchymal papilla, grows in cycles giving rise to a new hair shaft during each cycle. The ability to cycle endows the follicle with regenerative properties. The evolution of hair follicle engineering began with the recognition in the early 1960's that hair follicles could be transplanted clinically into a foreign site and still grow a shaft typical of the donor site. Since that time, it has been found that the follicular papilla has hair follicle inducing properties and that the hair follicle houses within it epithelial stem cells that can respond to hair inductive signals. These findings have laid the foundation for isolating hair-forming cells, for expanding the cells in culture, and for forming new follicles in vivo.

Stearoyl-coenzyme A (CoA) desaturase (SCD) is a key enzyme involved in the conversion of saturated fatty acids into monounsaturated fatty acids. Previously, two members of this gene family, namely, Scd1 and Scd2, have been reported. Here we report the identification and characterization of a novel member of this family, Scd3, whose expression is restricted to mouse skin, specifically to the sebaceous gland. The Scd3 gene codes for a transcript of approximately 4.9 kb with an open reading frame that results in a 359-amino-acid protein. Scd3 shares 91 and 88% identity in the protein-coding region with Scd1 and Scd2, respectively, and maps to mouse chromosome 19 in very close proximity to Scd1 and Scd2. Unlike Scd1, Scd3 expression is higher in male mouse skin than in female mouse skin. The promoter sequence of Scd3 reveals similarity with Scd1 in the proximal region but also possesses several distinctive features including the polyunsaturated fatty acid-response element. Scd3 is expressed in the skin of young asebia mutant mice (Scd1(ab2J)/Scd1(ab2J)) in the absence of Scd1. Scd3 expression changes during the mouse hair cycle but not as dramatically as Scd1. The tissue-specific and sex-dependent expression of Scd3 suggests the presence of gene- and hormonal-specific control mechanisms.

A critical step in the synthesis of unsaturated fatty acids is catalysed by stearoyl-CoA desaturase (Scd). To determine the regulation of human Scd, we characterized the gene and its transcripts. Screening a human keratinocyte cDNA library and analysis of 3ʹ-RACE (rapid amplification of cDNA ends) products from various tissues yielded a 5.2 kb cDNA encoding a 359 amino acid protein with a calculated molecular mass of 41.5 kDa. Analysis of 3ʹ-RACE products suggested that alternative usage of polyadenylation sites generates two transcripts of 3.9 and 5.2 kb, a result consistent with Northern analysis. Southern analysis demonstrated the existance of two SCD loci in the human genome. Chromosomal mapping localized one locus to chromosome 10, and the second locus to chromosome 17. Characterization of genomic clones isolated from chromosome-specific libraries revealed that only the locus on chromosome 10 contained introns. Sequence analysis of the intron-less locus displayed multiple nucleotide insertions and deletions, as well as in-frame stop codons. Reverse transcriptase-PCR analysis performed with primers specific to the intron-less locus failed to produce a PCR product from brain, liver and skin RNA, indicating that the locus on chromosome 17 is most likely a transcriptionally inactive, fully processed pseudogene. These results suggest strongly that there is one structural SCD gene in the human genome, and that it generates two transcripts by use of alternative polyadenyation sites. Although the primary sequence and intron-exon structure of SCD is phylogenetically conserved, divergence between rodent and human is seen in the number of SCD genes and in the generation of alternative transcripts, suggesting a species-specific component of SCD regulation and function.

A search for new genes was performed in a 220-kb region around the tumor necrosis factor gene cluster in the human central major histocompatibility complex region using a cDNA hybridization and selection method. In addition to the seven known genes in this region, we identified a new gene that is preferentially expressed in spleen. We also identified two pseudogenes that have high degrees of homology to cytokeratin and cyclophillin, respectively. Expressed sequences for a human homologue of the mouse B144 gene were also found in the current analysis. RT-PCR analysis showed that B144 is expressed in spleen, in thymus, and prominently in the macrophage cell line, U937. We also independently identified the BAT1 gene to be the well-conserved homologue of a previously described rat liver nuclear protein.

To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex. These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F. In addition to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by us and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region. We also identified a long repetitive DNA element in the region between HLA-B and HLA-E. Homologues of this element were located at several sites in the human genome outside of the HLA complex. The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions. Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.

Identification of transcribed sequences by cDNA selection is a potentially rapid and efficient way of scanning large genomic DNA fragments for the presence of genes. To evaluate this approach further, we have applied it to three yeast artificial chromosomes (YACs) and examined the products obtained from a total of about 1100 kb from two regions of the human major histocompatibility complex (MHC). One YAC was derived from an extensively studied portion of the Class II region of the MHC. The cDNAs recovered from this YAC included representatives of the previously described genes as well as one or more cDNA clones not described in the databases. A second YAC spanned about 330 kb of DNA surrounding the Class I gene HLA-A. In addition to Class I clones, 10 distinct cDNA products were identified from this YAC. A third YAC contained about 700 kb of human DNA, including 260 kb of overlap with the second YAC, and recovered an additional cDNA complementary to YAC B30 H3 DNA. Overall, the method is shown to be able to detect very scarce cDNAs and to detect a large fraction of coding sequences in YAC clones. Advantages and limitations of the approach are discussed.

We describe the construction and characterization of methylation-resistant sequence-tagged Not I linking clones specific for the X chromosome, referred to as Not I- Bsu E linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with Bsu E methylase (M. Bsu E), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant Not I sites by insertion of a kanamycin-resistance gene in the clones cleavable by Not I. The frequent occurrence of Not I sites in CpG islands is expected to cause methylation of a large number of Not I sites with Bsu E methylase, thereby rendering them resistant to Not I cleavage. Thus, the combination of M. Bsu E and Not I yields less frequent cutting than the Not I alone. We have isolated, partially squenced, and characterized 113 Not I- Bsu E linking clones, and mapped 50 clones to various regions along the chromosome.

We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.

We have refined the position of asebia locus by genotyping DNA from more than 600 backcross mice derived from asebia mouse and a genetically unrelated strain. One of the candidate genes in the locus is stearoyl-CoA desaturase (SCD). Previously two members of this gene family, namely SCD1 and SCD2, have been described. We have found, for the first time, that these SCD genes are expressed in skin. Moreover, we have identified a third species of SCD in the mouse skin. The most prominent SCD species is SCD1 in the mouse skin. The implications of this gene family to skin are discussed.

Pattern recognition is at the heart of clinical dermatology and dermato- pathology. Yet, while every practitioner of the art of dermatological diagnosis recognizes the supreme value of diagnostic cues provided by defined patterns of 'efflorescences', few contemplate on the biological basis of pattern formation in and of skin lesions. Vice versa, developmental and theoretical biologists, who would be best prepared to study skin lesion patterns, are lamentably slow to discover this field as a uniquely instructive testing ground for probing theoretical concepts on pattern generation in the human system. As a result, we have at best scraped the surface of understanding the biological basis of pattern formation of skin lesions, and widely open questions dominate over definitive answer. As a sym- metry-breaking force, pattern formation represents one of the most fundamental principles that nature enlists for system organization. Thus, the peculiar and often characteristic arrangements that skin lesions display provide a unique opportunity to reflect upon - and to experimentally dissect - the powerful organizing principles at the crossroads of developmental, skin and theoretical biology, genetics, and clin- ical dermatology that underlie these - increasingly less enigmatic - phenomena. The current 'Controversies' feature offers a range of different perspectives on how pattern formation of skin lesions can be approached. With this, we hope to encour- age more systematic interdisciplinary research efforts geared at unraveling the many unsolved, yet utterly fascinating mysteries of dermatological pattern forma- tion. In short: never a dull pattern!

Hair follicle formation and cycling involve extensive and continuous interactions between epithelial and mesenchymal components. A system for rapidly and reproducibly generating hair follicles from dissociated epithelial and mesenchymal cells is described here. The system serves both as a tool for measuring the trichogenic property of cells and as a tool for studying the mechanisms that dissociated cells use to assemble an organ. In this system, hair follicles develop when dissociated cells, isolated from newborn mouse skin, are injected into adult mouse truncal skin. This morphogenetic process involves the aggregation of epithelial cells to form clusters that are sculpted by apoptosis to generate "infundibular cysts". From the "infundibular cysts", hair germs form centrifugally followed by follicular buds and then pegs that grow asymmetrically to differentiate into cycling mature pilosebaceous structures. Marker studies correlate the molecular differentiation of these follicles with in situ systems. This study suggests that the earliest phase of a developing epithelial-mesenchymal system--even from dissociated cell preparations--requires an epithelial platform.

A spontaneous, autosomal, recessive mouse mutation exhibiting mild scaly skin, progressive scarring alopecia, slightly runted growth, and photophobia arose at The Jackson Laboratory in 1993 in the inbred mouse strain DBA/1LacJ. Because this mutant mouse showed genetic, anatomical, and laboratory similarities to the asebia mutation, crosses were done between the new mutant and mice carrying the asebia-J allele. Because the F1 offspring were affected, indicating the two mutants were allelic, the new mutation was named asebia-2J. Careful histological analysis of skin development of mice homozygous and heterozygous for either asebia-J or asebia-2J revealed that both types of mutant mice are very similar regardless of their background. Notable histopathological features of mice homozygous for either allele included extreme sebaceous gland hypoplasia, abnormally long anagen follicles, retained inner root sheath, hair fiber perforation of the anagen follicle base, and progressive follicular replacement by scarring. In this article we present a new pathogenetic hypothesis based on the importance of the sebaceous gland in hair fiber sheath dissociation: in the absence of a functional sebaceous gland the hair follicle is destroyed. The cutaneous pathology of this mutant mouse underscores the importance of the sebaceous gland to follicular biology and presents an animal model for studying the human scarring alopecias, which characteristically begin with sebaceous gland ablation.

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes, including conotruncal heart defects, cleft palate, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Both syndromes are thought to be the result of a developmental field defect. Using two independent gene-isolation procedures, we isolated a new catenin family member termed ARVCF (armadillo repeat gene deleted in VCFS) from the interval deleted in VCFS. ARVCF encodes a protein of 962 amino acids that contains a coiled coil domain and 10 tandem armadillo repeats. The primary structure of the protein is most closely related to the murine catenin p120CAS, which suggests a role for ARVCF in protein-protein interactions at adherens junctions. ARVCF is expressed ubiquitously in all fetal and adult tissues examined. This gene is hemizygous in all VCFS patients with interstitial deletions. Based on the physical location and potential functions of ARVCF, we suggest that hemizygosity at this locus may play a role in the etiology of some of the phenotypes associated with VCFS.

We have applied cDNA hybridization selection to nine YACs spanning 3 Mb of genomic DNA from a region centromeric to HLA-A to the histone cluster that lies telomeric to the human major histocompatibility complex (MHC). In addition to Class I genes and pseudogenes, we describe over 63 genes and 23 additional expressed sequence tags distributed throughout the region. Many of the full-length genes belong to gene families. Prominent among these are a group of genes encoding proteins showing homology to the carboxyl-terminal sequences of butyrophilin and an additional group of zinc finger genes. We also detected several previously undefined genes that are specifically expressed in cells of the immune system, indicating a more complex role of the MHC in the immune response than has been appreciated.

The murine major histocompatibility complex (MHC) includes sequences that are responsible for haplotype-specific odor types that, in turn, influence mating preference. We report that there are several olfactory receptor genes or pseudogenes in the Class I region of the human MHC. At least one of these genes is intact, appears to encode an mRNA, and is quite homologous to a previously reported murine olfactory receptor.

We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.

A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E. coli. Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum-mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum-mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.