Suprabasal marker proteins distinguishing keratinizing squamous epithelia: Cytokeratin 2 polypeptides of oral masticatory epithelium and epidermis are different

Terminal differentiation of squamous epithelia is usually characterized by the synthesis of a subset of cytokeratins (CKs) in suprabasal cell layers which become major components of the intermediate filament (IF) bundle cytoskeleton of the maturing cells. We have examined the significance, molecular nature and pattern of synthesis of the elusive human CK 2 by analyzing mRNAs from certain stratified epithelia, using in vitro translation, cDNA cloning. Northern blotting and in situ hybridization. We show that genuine polypeptides with the typical gel electrophoretic mobility of CK 2 exist but that the CK 2 present in the masticatory epithelia of hard palate and gingiva (CK 2p) differs from that found in epidermis (CK 2e) by its amino acid sequence and is encoded by a different gene. The two CKs 2 show only limited sequence homology (71% identical amino acid positions in the rod domain), and the oral CK 2p is more closely related to the corneal CK 3 (86%), as is also indicated by the cross-reaction of monoclonal antibody AE5. By in situ hybridization and immunocytochemistry, we further show that both CK 2e and CK 2p are expressed only in suprabasal cell layers of the specific epithelia where they can accumulate to represent major cytoskeletal proteins. We discuss this tissue-type specificity of CK 2 synthesis in otherwise morphologically and biochemically similar epithelia in relation to differences of IF appearance and packing in upper strata between epidermal and masticatory epithelia as well as to tissue formation and differentiation during development.     

Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements.

Genomic 17 beta-hydroxysteroid-dehydrogenase (17-HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17-HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17-HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17-HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5' flanking regions contained sequences similar to the consensus sequences of cis-acting elements, defined as regulators of 17-HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid-response elements and a cyclic-AMP regulatory element.     

Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were approximately 20-30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of approximately 2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-alpha. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.     

棉铃虫细胞色素P450 CYP9A12基因5′-上游区的克隆及序列分析

细胞色素P450基因CYP9A12的过量表达已被证实与棉铃虫Helicoverpa armigera对拟除虫菊酯的抗性相关。为探明棉铃虫CYP9A12基因的表达调控机理,根据棉铃虫CYP9A12基因cDNA全长的5′-末端核苷酸序列,采用基因组步移方法,获得CYP9A12的5′-上游区序列（总长为3 575 bp）。与cDNA序列进行比对,表明在起始密码子上游3 bp处有一长为2 124 bp的内含子。利用NNPP分析软件预测出转录起始位点,与根据CYP9A12全长cDNA序列推测的结果是一致的。TFSEARCH 1.3软件分析转录因子结合位点的结果显示,该序列不仅包含启动子的核心结构序列——TATA-box和CAAT-box,亦包含多个转录因子结合位点,如GATA-1,CdxA,Dfd等。本研究结果为深入研究棉铃虫CYP9A12的表达调控机制及其参与杀虫剂抗性的分子机理奠定了一定基础。     

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.     

Regulation of basal and luminal cell-specific cytokeratin expression in rat accessory sex organs. Evidence for a new class of androgen-repressed genes and insight into their pairwise control.

Co-expression of cytokeratin (CK) pairs has been found to be associated with specific epithelial cell types whose expressions are developmentally regulated. In the prostate, CK 8 and 18 have been identified as luminal cell-specific markers, and CK 5 and 15 have been identified as basal cell-specific markers. In this study, we report the cloning and sequencing of a full-length CK 8 cDNA (1.9 kilobases) from a rat ventral prostate (VP) cDNA library. Although the open reading frame shares 90% homology with mouse CK 8 sequences, nucleotide comparison revealed that rat CK 8 cDNA comprises a species-specific sequence on both 5' and 3' ends. The steady-state levels of CK 8 mRNA were elevated in VP, seminal vesicle (SV), and liver of a castrated rat but not in the other organs such as the coagulating gland, bladder, and thymus. Unlike the other androgen-repressed genes, elevated CK 8 mRNA levels persisted even after the glandular involution was completed, indicating that CK 8 is a new class of androgen-repressed gene. The regression of CK 8 expression may be androgen receptor-mediated, since androgen but not estrogen administration to castrated hosts repressed the CK 8 mRNA levels, and this effect can be antagonized by the simultaneous administration of an antiandrogen (4-hydroxyflutamide). Immunohistochemical staining of prostatic tissues reveals that the CK 8 filamentous structure is shifted reversibly from a uniform distribution to a predominantly basal surface upon androgen deprivation. We noted that the steady-state levels of CK 8 protein remain rather constant throughout the various hormonal treatment, and the steady-state levels of CK 8 mRNA and the rate of CK 8 protein synthesis are consistently elevated. These results suggest that the turnover rate of CK 8 protein may be elevated in the prostatic epithelium from the castrated host. Similarly, the steady-state levels of CK 15 and 18 mRNA in VP and SV are also repressed in an androgen-dependent manner. These data, taken together, indicate that pairwise control of luminal (and possibly basal) specific cytokeratin gene expression remains intact in both VP and SV tissues and that the levels of CK mRNAs expression are negatively regulated by androgen.     

Sequence and expression of a type II keratin, K5, in human epidermal cells.

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

A novel Xenopus laevis larval keratin gene, xlk2: its gene structure and expression during regeneration and metamorphosis of limb and tail

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the "de-differentiated" basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.     

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.     

Isolation of the feline alpha1,3-galactosyltransferase gene, expression in transfected human cells and its phylogenetic analysis

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.