Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA(Tyr) is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.     

Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion

A partial physical map has been constructed of the human class Mu glutathione S-transferase genes on chromosome 1p13.3. The glutathione S-transferase genes in this cluster are spaced about 20 kilobase pairs (kb) apart, and arranged as 5'-GSTM4-GSTM2-GSTM1-GSTM5-3'. This map has been used to localize the end points of the polymorphic GSTM1 deletion. The left repeated region is 5 kb downstream from the 3'-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region is 5 kb downstream from the 3'-end of the GSTM1 and 10 kb upstream from the 5'-end of the GSTM5 gene. The GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb HindIII fragments. The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most GSTM1-0 deletions involve recombinations between the same two regions. We have cloned and sequenced the deletion junction that is produced at the GSTM1-null locus; the 5'- and 3'-flanking regions are more than 99% identical to each other and to the deletion junction sequence over 2.3 kb. Because of the high sequence identity between the left repeat, right repeat, and deletion junction regions, the crossing over cannot be localized within the 2.3-kb region. The 2.3-kb repeated region contains a reverse class IV Alu repetitive element near one end of the repeat.     

An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells

Central nervous system (CNS) progenitor cells transiently proliferate in the embryonic neural tube and give rise to neurons and glial cells. A characteristic feature of the CNS progenitor cells is expression of the intermediate filament nestin and it was previously shown that the rat nestin second intron functions as an enhancer, directing gene expression to CNS progenitor cells. In this report we characterize the nestin enhancer in further detail. Cloning and sequence analysis of the rat and human nestin second introns revealed local domains of high sequence similarity in the 3' portion of the introns. Transgenic mice were generated with the most conserved 714 bp in the 3' portion of the intron, or with the complete, 1852 bp, human second intron, coupled to the reporter gene lacZ. The two constructs gave a very similar nestin-like expression pattern, indicating that the important control elements reside in the 714 bp element. Expression was observed starting in embryonic day (E)7.5 neural plate, and at E10.5 CNS progenitor cells throughout the neural tube expressed lacZ. At E12.5, lacZ expression was more restricted and confined to proliferating regions in the neural tube. An interesting difference, compared to the rat nestin second intron, was that the human intron at E10.5 mediated lacZ expression also in early migrating neural crest cells, which is a site of endogenous nestin expression. In conclusion, these data show that a relatively short, evolutionarily conserved region is sufficient to control gene expression in CNS progenitor cells, but that the same region differs between rodents and primates in its capacity to control expression in neural crest cells.     

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.     

The structure and organization of the human NPAT gene

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.     

Self-organization of binocular disparity tuning by reciprocal corticogeniculate interactions

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3' untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3' untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' exon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (> 36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. The 5' regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIA gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C-->A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2 + 11A/G, IVS4 + 44C/A, and IVS6 + 538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7 (CA)10-11 CCCCCTATA (TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene, HFE, were examined and found to be normal. One hemochromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromatosis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.     

Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5' splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5' splice site and by an exonic enhancer differ mechanistically.