Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Class II genes of the human major histocompatibility complex. Comparisons of the DQ and DX alpha and beta genes

The human major histocompatibility complex, HLA, contains the genes of several class II molecules. We present here the molecular maps of the DQ and DX subregions and analyze the sequences of the polymorphic DQ alpha and DQ beta genes as well as the DX alpha and DX beta genes. The DQ alpha and DQ beta genes are oriented in opposite directions, approximately 12 kilobases apart. The DX alpha and DX beta genes are similarly oriented about 8 kilobases. The exon-intron organizations of the DQ alpha and DX alpha genes are analogous to those of other class II alpha genes. Comparison of the DQ alpha gene sequence to three DQ alpha cDNA clones shows that amino acid replacements are predominantly located between residues 45 and 80 in the amino-terminal domain. Analysis of the frequency of silent and replacement substitutions indicates that there is little selection against replacements in DQ alpha first domains. The exons encoding the second domains of DQ alpha and DX alpha are virtually identical, suggesting that a gene conversion event has occurred between these genes. The DX beta gene is very similar to the DQ beta gene but differs in the cytoplasmic portion. The DX beta gene contains a separate exon of 24 nucleotides encoding the core of the cytoplasmic tail. This exon is not expressed in the DQ beta genes due to a nonfunctional splice junction. Comparison of the number of nucleotide substitutions in the DQ beta first and second domain exons suggests that little or no phenotypic selection acts on the first domain whereas the second domain is under strong selection.     

Structure of the murine immune response I-A beta locus: sequence of the I-A beta gene and an adjacent beta-chain second domain exon

The murine major histocompatibility complex I region encodes two class II antigens, I-A and I-E. From a mouse spleen DNA cosmid library of the b haplotype, we isolated a clone containing the entire I-A beta gene and a separate exon encoding a beta-chain second domain (A beta 2). The A beta gene, encompassing more than 6 kb, is encoded by six exons corresponding to the different domains of the A beta polypeptide. The translated A beta amino acid sequence displays 73% homology to human DC beta chains; homologies to other subsets of human beta chains are lower, establishing that I-A corresponds structurally to DC. The A beta 2 exon is about 20 kb centromeric to the A beta gene. Its translated amino acid sequence includes all the conserved amino acids of other class II beta-chain second domains. It shows about 60% homology to each of three subsets of human beta chains available for comparison, and to the A beta chain. No A beta 2 first domain exon has been detected with A beta or DC beta probes.     

Exon structure of a mannose-binding protein gene reflects its evolutionary relationship to the asialoglycoprotein receptor and nonfibrillar collagens

Cloned cDNAs encoding mannose-binding proteins isolated from rat liver have been used to isolate one of the genes encoding this group of proteins. This gene, which encodes the minor form of binding protein (designated MBP-A), has been characterized by sequence analysis. The protein-coding portion of the mRNA for the MBP-A is encoded by four exons separated by three introns. The NH2-terminal, collagen-like portion of the protein is encoded by the first two exons. These exons resemble the exons found in the genes for nonfibrillar collagens in that the intron which divides them is inserted between the first two bases of a glycine codon and the exons do not have the 54- or 108-base pair lengths characteristic of fibrillar collagen genes. The carbohydrate-binding portion of MBP-A is encoded by the remaining two exons. This portion of the protein is homologous to the carbohydrate-recognition domain of the hepatic asialoglycoprotein receptor, which is encoded by four exons. It appears that the three COOH-terminal exons of the asialoglycoprotein receptor gene have been fused into a single exon in the MBP-A gene. The organization of the MBP-A gene is very similar to the arrangement of the gene encoding the highly homologous pulmonary surfactant apoprotein, although one of the intron positions is shifted by a single amino acid. The 3' end of a mannose-binding protein pseudogene has also been characterized.     

Structure of the Rat Aromatic L-Amino Acid Decarboxylase Gene: Evidence for an Alternative Promoter Usage

Abstract: Aromatic L-amino acid decarboxylase catalyzes the biosynthesis of the neurotransmitters dopamine and serotonin. This enzyme is also expressed in nonneuronal tissues. Two reported cDNA sequences show that the pheochromocytoma message differs from the liver message only at the 5'untranslated region. We present the complete exonal organization and promoter sequences of the rat gene encoding this enzyme. The rat aromatic L-amino acid decarboxylase gene is composed of two promoters and 16 exons spanning more than 80 kb in the genome. The first exon carries the majority of the 5'untranslated sequence of the liver cDNA, and the second exon carries that of the pheochromocytoma cDNA. In the third exon, there are two alternatively utilized splicing acceptors specific to the first and second exons. Therefore, both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. The sequence of each promoter region shows putative binding sites for octamer factors and AP-2.     

Structure, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene

We have determined the nucleotide sequence and genomic organization of the mouse Lyt-2 T lymphocyte differentiation antigen gene. This gene consists of five exons and four introns, and the organization roughly parallels the protein domains. Alternative splicing to include or exclude exon IV (encoding part of the cytoplasmic tail) results in two forms of mRNA and accounts for the difference in size between the alpha- and alpha'-chains of Lyt-2. The gene structure provides further evidence for the evolutionary relationship between Lyt-2 and immunoglobulin genes. Comparison of the nucleotide sequence of the Lyt-2.1 and Lyt-2.2 alleles shows a high degree of conservation, but indicates that a single nucleotide change and consequent amino acid substitution in the variable region-like domain accounts for the serologic difference between these two alleles.     

Molecular cloning of a gene for a member of carcinoembryonic antigen (CEA) gene family; signal peptide and N-terminal domain sequences of nonspecific crossreacting antigen (NCA)

A 2073-base pair DNA fragment containing a part of gene for a member of carcinoembryonic antigen (CEA) gene family, has been isolated from human DNA library after screening with 32P-labeled 53-mer oligodeoxyribonucleotide corresponding to N-terminal 18 amino acids of CEA gene family and cDNA encoding CEA (1,2). The fragment contains two exons; the one encodes the first 60% of signal peptide and the other the rest of it in addition to 107 amino acids which correspond to the N-terminal domain of CEA (1,2). Apparently, the second intron is inserted between the first and the second nucleotides of the codon for 108th amino acid. The presence of Ala instead of Val as the 21st amino acid of the N-terminal domain indicates that the exon encodes nonspecific crossreacting antigen (NCA).     

Characterization of the gene encoding the major rat liver asialoglycoprotein receptor

A cloned cDNA encoding the major rat liver asialoglycoprotein receptor has been used to analyze the gene for this protein. Genomic Southern blot analysis reveals that the gene is contained on a single EcoRI restriction fragment and is unique. A clone containing the gene (isolated from a rat liver genomic library) has been characterized by sequence analysis. The mRNA for the receptor is encoded by nine exons separated by eight introns. The first exon is confined to the 5'-untranslated region of the mRNA, the second exon encodes most of the cytoplasmic NH2-terminal domain of the receptor polypeptide, the third exon corresponds to the hydrophobic transmembrane portion of the polypeptide, and the remaining exons encode the extracellular parts of the receptor. Some, but not all, of the divisions between exons correspond to boundaries between functional domains of the polypeptide.     

Concerted and divergent evolution within the rat gamma-crystallin gene family

The nucleotide sequences of six rat gamma-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5' non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3' non-coding region. In the gamma 1-2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 X 10(3) to 1.9 X 10(3) base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a' of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b') is constant. We postulate that this variability in region a' is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a' is now preserved. Hence, it might specify the specific functional property of each gamma-crystallin protein within the lens.     

Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells.

We have determined the nucleotide sequences of Amy32b, a type A alpha-amylase gene, and of the gene for aleurain, a thiol protease closely related to mammalian cathepsin H. Both are expressed in barley aleurone cells under control of the plant hormones gibberellic acid and abscisic acid, but only aleurain is expressed at high levels in other barley tissues. Sequence analysis indicates that the 5' end of the aleurain gene, comprising 3 exons and 2 introns, may have become associated with the remainder of the gene, encoding the protease domain of the protein, by some sort of recombination event. This 5' domain of the gene is very G + C-rich and is flanked by inverted repetitive sequences. We found two different groups of homologous sequence elements. The first group consists of four blocks of sequences conserved in the same spatial arrangement in both genes; these are arranged at similar intervals upstream from the Amy32b TATA box and from a TATA box present in intron 3 of aleurain, outside of the 5' domain and upstream from the protease domain. A part of two of these conserved sequences is similar to the core sequence of certain enhancer elements characterized from mammalian cells. The second group of homologous elements is present in the upstream region of both genes. We speculate that these conserved sets of sequences may have some role in either the tissue specificity of expression of the genes or in some part of the hormonal regulation imposed on them.     

Chromosomal Location and Some Structural Features of Human Clathrin Light-Chain Genes (CLTA and CLTB)

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.     

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.