Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region.

Human alpha-galactosidase A (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) is a lysosomal hydrolase encoded by a gene localized to the chromosomal region Xq22. The deficient activity of this enzyme results in Fabry disease, an X chromosome-linked recessive disorder that leads to premature death in affected males. For studies of the structure and function of alpha-galactosidase A and for characterization of the genetic lesions in families with Fabry disease, the full-length cDNA was isolated, sequenced, and used to screen human genomic libraries. The 1393-base-pair full-length cDNA had a 60-nucleotide 5' untranslated region and encoded a precursor peptide of 429 amino acids including a signal peptide of 31 residues. Three overlapping lambda clones spanning 32 kilobases were identified that contained the entire approximately equal to 12-kilobase chromosomal gene as well as approximately equal to 9 and approximately equal to 11 kilobases of 5' and 3' flanking sequence, respectively. The gene had seven exons. The genomic exonic and full-length cDNA sequences were identical. All intron-exon splice junctions conformed to the GT/AT consensus sequence. The 5' flanking region of this lysosomal housekeeping gene contained Sp1 and CCAAT box promoter elements as well as sequences corresponding to the activator protein 1 (AP1), octanucleotide ("OCTA"), and "core" enhancer elements. There was an upstream "HTF" island (Hpa II tiny fragments) followed by four direct repeats of the "chorion box" enhancer. The unique lack of a 3' untranslated sequence in the alpha-galactosidase A cDNA was confirmed by sequencing additional cDNA clones and the genomic 3' region.     

Complete primary structures of two major murine serum amyloid A proteins deduced from cDNA sequences.

cDNA clones encoding two major mouse serum amyloid A proteins, SAA1 and SAA2, were isolated from a liver cDNA library of the lipopolysaccharide-stimulated BALB/c mouse, and their nucleotide sequences were determined. The insert of the SAA2 cDNA clone contained 607 nucleotides with a 5' untranslated region of 36 nucleotides, a signal peptide region corresponding to 19 amino acids, a mature protein region corresponding to 103 amino acids, and a 3' untranslated region of 202 nucleotides. The SAA1 cDNA insert contained 549 nucleotides specifying a part of a signal peptide region, a mature protein region, and a 3' untranslated region. A comparison of the nucleotide and deduced amino acid sequences of SAA1 cDNA with that of SAA2 cDNA showed a high degree of homology: 95% nucleotide sequence homology in the coding region (91% amino acid sequence homology) and 90% homology in the 3' untranslated region. One of nine amino acid differences between SAA1 and SAA2 predicted from the cDNA sequences was located in a putative proteolytic cleavage site for amyloid A protein formation: SAA2 had the Thr-Met sequence in this site, while SAA1 had the Thr-Ile sequence. This suggests that SAA1, which does not deposit as amyloid A protein, is also potentially susceptible to putative proteolytic enzymes. In addition, as compared with mouse SAA2, human SAA1, monkey and mink amyloid A protein, mouse SAA1 had two unique substitutions, which may play a role in differential deposition of mouse SAA isotypes in amyloid tissues.     

Molecular basis of human growth hormone gene deletions.

Crossover sites resulting from unequal recombination within the human growth hormone (GH) gene cluster that cause GH1 gene deletions and isolated GH deficiency type 1A were localized in nine patients. In eight unrelated subjects homozygous for 6.7-kilobase (kb) deletions, the breakpoints are within two blocks of highly homologous DNA sequences that lie 5' and 3' to the GH1 gene. In seven of these eight cases, the breakpoints map within a 1250-base-pair (bp) region composed of 300-bp Alu sequences of 86% homology and flanking non-Alu sequences that are 600 and 300 bp in length and are of 96% and 88% homology, respectively. In the eighth patient, the breakpoints are 5' to these Alu repeats and are most likely within a 700-bp region of 96% homologous DNA sequences. In the ninth patient homozygous for a 7.6-kb deletion, the breakpoints are contained within a 29-bp perfect repeat lying 5' to GH1 and the human chorionic somatomammotropin pseudogene (CSHP1). Together, these results indicate that the presence of highly homologous DNA sequences flanking GH1 predispose to recurrent unequal recombinational events presumably through chromosomal misalignment.     

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.     

Complete primary structures of the E beta chain and gene of the mouse major histocompatibility complex.

Using the cross-hybridization with plasmid pDC beta-1, containing the cDNA coding for the DC beta chain of the human major histocompatibility complex class II molecules, we have cloned and subjected to sequence analysis both the cDNA and genomic gene for the E beta chain of the BALB/c (d haplotype) mouse. The nucleotide sequences of the cDNA and genomic DNA clones permitted us to deduce the entire primary structure of the E beta chain and the complete exon-intron structure of the E beta gene. Unlike alpha chain genes that contain five exons, the E beta gene consists of six exons corresponding to the six functional domains--the leader, beta 1 and beta 2 domains, transmembrane peptide, intracytoplasmic peptide, and 3' untranslated region. In addition, two short blocks of sequences common to alpha and beta chain genes were identified in the 5' flanking regions. We propose that these sequences are involved in the coordinate expression of alpha and beta chains.     

Structural organization of glycophorin A and B genes: glycophorin B gene evolved by homologous recombination at Alu repeat sequences.

Glycophorins A (GPA) and B (GPB) are two major sialoglycoproteins of the human erythrocyte membrane. Here we present a comparison of the genomic structures of GPA and GPB developed by analyzing DNA clones isolated from a K562 genomic library. Nucleotide sequences of exon-intron junctions and 5' and 3' flanking sequences revealed that the GPA and GPB genes consist of 7 and 5 exons, respectively, and both genes have greater than 95% identical sequence from the 5' flanking region to the region approximately 1 kilobase downstream from the exon encoding the transmembrane regions. In this homologous part of the genes, GPB lacks one exon due to a point mutation at the 5' splicing site of the third intron, which inactivates the 5' cleavage event of splicing and leads to ligation of the second to the fourth exon. Following these very homologous sequences, the genomic sequences for GPA and GPB diverge significantly and no homology can be detected in their 3' end sequences. The transition site from homologous to nonhomologous sequences can be localized within Alu repeat sequences. The analysis of the Alu sequences and their flanking direct repeat sequences suggest that an ancestral genomic structure has been maintained in the GPA gene, whereas the GPB gene has arisen from the acquisition of 3' sequences different from those of the GPA gene by homologous recombination at the Alu repeats during or after gene duplication.     

Soybean leghemoglobin gene family: normal, pseudo, and truncated genes.

Leghemoglobin (Lb) genes in soybean represent a small family of closely related genes. Three Lb sequences isolated from a genomic library were analyzed at the nucleotide sequence level. A Lb gene present on an 11.5-kilobase (kb) EcoRI genomic fragment spans approximately 1,200 nucleotides and is interrupted at amino acid positions 32 to 33, 68 to 69, and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain the consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the DNA from the recombinant phage containing the 11.5-kb EcoRI genomic fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequence of the second gene showed that this gene is incomplete, containing only exons 3 and 4. The deduced amino acid sequence of this gene, although showing 76% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present on a second genomic clone containing a 4.2-kb EcoRI fragment revealed a truncated Lb gene showing homology with the last exon and the noncoding region at the 3' end of the two other Lb genes.     

Complete cDNA and derived amino acid sequence of human factor V.

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.     

Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus.

We have isolated cDNA clones of the human c-myb mRNA that contain approximately 3.4 kilobases of the approximately 3.8-kilobase mRNA sequence. Nucleotide sequence analysis shows that the c-myb mRNA contains an open reading frame of 1920 nucleotides, which could encode a 72-kDa protein. The cDNA nucleotide sequence and the predicted amino acid sequence of the c-myb protein are highly homologous to the corresponding chicken and mouse proteins. In particular, a region toward the NH2 terminus of the protein containing a 3-fold tandem repeat of 51 residues is evolutionarily conserved and is the only region of homology with the Drosophila c-myb protein. This region may represent a functionally important structure, most likely the DNA-binding domain. cDNA clones have been used to isolate genomic clones and to define a preliminary intron/exon organization of the c-myb gene. Identification of 5' and 3' coding and noncoding exons indicates that the human c-myb locus spans a 40-kilobase region.     

Comparison of mouse immunoglobulin gamma 2a and gamma 2b chain genes suggests that exons can be exchanged between genes in a multigenic family.

A 23-kilobase EcoRI DNA fragment coding for the BALB/c immunoglobulin gamma 2a chain was cloned from mouse embryo DNA in the cosmid pJC74, and a nucleotide sequence of 1904 bases was determined for the entire constant region (CH1, CH2, and CH3), the three intervening sequences (IVS 1, IVS 2, and IVS 3) and the 5' and 3' flanking sequences. When the gamma 2a chain nucleotide sequence was compared with the gamma 2b chain nucleotide sequence, the percent homology of corresponding segments (excluding deletion and insertion) was 82% for the 5' flanking sequence, 87% for CH1, 84% for IVS 1, 96% for the hinge, 95% for IVS 2, 94.6% for CH2, 86% for IVS 3, 74% for CH3, 89% for the 3' untranslated region, and 92% for the 3' flanking region. These findings show that different domains of gamma 2a and gamma 2b have independent rates of evolution and that some of the noncoding segments of the gene are more conserved than are adjacent coding segments. Hypotheses on the possible role of IVS is gene evolution and expression are discussed.     

Structure of the human interleukin 2 gene.

We have cloned two species of EcoRI-cleaved DNA segments that together cover the entire sequence for the human interleukin 2 gene and have determined the nucleotide sequence of the gene and its flanking regions. The gene contains three introns and the exon sequences can be aligned with the previously reported cDNA sequence almost perfectly except for a few nucleotides in the 3' nontranslated region. The promoter region contains a prototype "TATA" sequence as well as a notable palindromic sequence. Particularly interesting is the presence of sequences in this region that are homologous to the promoter region of the human interferon-gamma gene. In addition, a sequence that closely resembles the core sequence for the viral enhancer elements has been found in the second intron. Such sequences may play a role in the expression of the interleukin 2 gene in lectin- or antigen-stimulated T lymphocytes.     

Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome.

The human precursor gene for vasoactive intestinal peptide (VIP) and PHM-27, a peptide that has an NH2-terminal histidine and COOH-terminal methionine amide and is closely related in sequence and activity to VIP, was detected with synthetic oligodeoxynucleotide probes. These specific hybridization segments were constructed according to the neuroblastoma VIP cDNA sequence and contained up to 39 bases. The gene structure was partly deduced by hybridization to synthetic oligodeoxynucleotide probes and partly by direct chemical nucleotide sequencing. Four exons were discovered thus far; among them are two short exons separated by a 0.75-kilobase DNA stretch, one encoding PHM-27 and the second encoding VIP (exons 1 and 2). Each of these two exons encodes both the hormone amino acid residues as well as the post-translational processing signal sequences. The 3' splice sites of the two exons contain an identical stretch of nine nucleotides. At the cDNA level, the 3' splice sites contain the same stretch of six nucleotides, which are identically spliced. The occurrence of VIP and PHM-27 coding sequences on two separate exons of the human genome and the homology of their 3' splice site may allow alternative RNA processing as discussed below.     

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.