Organization, structure, and polymorphisms of the human profilaggrin gene

Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.     

Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes.

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other beta-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5' untranslated regions. We have previously reported that the 3' untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5' flanking region, 5' untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of beta-actin gene expression.     

Dynamics of actin evolution in dinoflagellates

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.     

A phylogenetic study of the palm family (Palmae) based on chloroplast DNA sequences from thetrnL —trnF region

Phylogenies of the palm family based on DNA sequences from thetrnL —trnF region of the chloroplast genome are presented. Although the region is highly conserved in palms and relatively few sites in the aligned data matrix are parsimony informative, a variety of relationships among members of the family are revealed by the analyses, some of which are congruent with the current classification of the palms, and others which are not. However, consensus trees contain high levels of ambiguosity, partly due to the inadequate numbers of informative characters in the dataset. Additional data are required before well resolved palm phylogenies can be generated.     

Characterization of 5‘—proximal sequence of mouse GABA transporter gene （GAT—1）

The cDNA molecule encoding the mouse GABA transporter gene(GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library.A positive clone,harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G,was fished out from the library,the 5‘ proximal region and intron 1 were sequenced and analysed,and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences.The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5‘ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay,and Southern-Western blot.The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.     

A repeat complex in the mitochondrial control region of Adélie penguins from Antarctica.

We have determined the nucleotide sequence of the entire mitochondrial control region (CR) of the Adélie penguin (Pygoscelis adeliae) from Antarctica. Like in most other birds, this CR region is flanked by the gene nad6 and transfer (t)RNA trnE(uuc) at the 5' end and the gene rns and trnF(gaa) at the 3' end. Sequence analysis shows that the Adelie penguin CR contains many elements in common with other CRs including the termination associated sequences (TAS), conserved F, E, D, and C boxes, the conserved sequence block (CSB)-1, as well as the putative light and heavy strand promoters sites (LSP-HSP). We report an extraordinarily long avian control region (1758 bp) which can be attributed to the presence, at the 3' peripheral domain, of five 81-bp repeat sequences, each containing a putative LSP-HSP, followed by 30 tetranucleotide microsatellite repeat sequences consisting of (dC-dA-dA-dA)30. The microsatellite and the 81-bp repeat reside in an area known to be transcribed in other species.