Murine Ia and human DR antigens: homology of amino-terminal sequences.

Murine Ia and human DR antigens were isolated and purified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis with allo- and xenoantisera, respectively. The I-A subregion antigen consists of two chains, designated Aalpha and Abeta, with molecular weights of 35,000 and 26,000, respectively. The I-C subregion antigen likewise consists of two chains, designated Calpha and Cbeta, with molecular weights of 32,000 and 29,000, respectively. Under nonreducing conditions, the Cbeta chain migrates appreciably more rapidly on sodium dodecyl sulfate/polyacrylamide gels than the reduced Cbeta chain, reflecting the presence of an intrachain disulfide bond. The human DR antigen is also a two-chain unit and contains DRalpha and DRbeta components with molecular weights of 34,000 and 28,000, respectively. The DRbeta chain migrates more rapidly before reduction than afterward, like the murine Cbeta chain. The DRbeta and Cbeta chains are also strikingly homologous if a single amino acid shift is imposed on one of those chains. Thus, human DR antigens strongly resemble the murine I-C subregion antigens.     

Brain-specific genes have identifier sequences in their introns.

The 82-nucleotide identifier (ID) sequence is present in the rat genome in 1-1.5 X 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. One brain-specific gene contains more than one ID sequence in its introns. There is an excess of ID sequences to brain genes, and some ID sequences appear to have been inserted as mobile elements into other genetic locations. Therefore, brain genes contain ID sequences in their introns, but not all ID sequences are located in brain gene introns. A brain ID consensus sequence has been obtained by comparing 8 ID nucleotide sequences.     

Molecular structure and chromosomal mapping of the human homolog of the agouti gene.

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.     

Molecular structure and evolutionary origin of human cardiac muscle actin gene.

Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.     

Mutated beta-actin gene: coexpression with an unmutated allele in a chemically transformed human fibroblast cell line.

By in vitro translation, we have identified the mRNA species that codes for a novel actin polypeptide (Ax-actin) in the chemically transformed human fibroblast line HuT-14. The relatedness of the coding sequences of the Ax- and beta-actin genes is indicated by our finding that pcDd actin ITL-I DNA, a recombinant plasmid DNA that contains a DNA sequence complementary to actin mRNA of Dictyostelium discoideum, hybridizes both the Ax-actin mRNA and the beta-actin mRNA but not the gamma-actin mRNA. In contrast, pcHa-1 DNA, a recombinant plasmid constructed by cloning a DNA sequence complementary to human actin mRNA from HuT-14 cells into pBR322, hybridized to all three mRNA species. In addition, no difference was observed between Ax- and beta-actin mRNAs when their molecular size was determined either by sucrose density gradient sedimentation or by methyl mercury agarose gel electrophoresis. Southern blot transfer of radioactive pcDd actin DNA to restriction endonuclease-digested Hut-14 DNA produced only a single hybrid band (a 6-kilobase fragment); the pcHa-1 DNA probe detected one additional band (a 3-kilobase fragment). These results suggest that HuT-14 cells contain only one copy per haploid genome for Ax- or beta-actin. When considered together with recent determination of the entire amino acid sequences of Ax- and beta-actin, our findings indicate that Ax-actin is the product of a mutated beta-actin gene and are evidence for the occurrence of a mutation in a chemically transformed cell.     

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Splicing of the ribosomal RNA precursor of Tetrahymena is an autocatalytic reaction, requiring no enzyme or other protein in vitro. The structure of the intervening sequence (IVS) appears to direct the cleavage/ligation reactions involved in pre-rRNA splicing and IVS cyclization. We have probed this structure by treating the linear excised IVS RNA under nondenaturing conditions with various single- and double-strand-specific nucleases and then mapping the cleavage sites by using sequencing gel electrophoresis. A computer program was then used to predict the lowest-free-energy secondary structure consistent with the nuclease cleavage data. The resulting structure is appealing in that the ends of the IVS are in proximity; thus, the IVS can help align the adjacent coding regions (exons) for ligation, and IVS cyclization can occur. The Tetrahymena IVS has several sequences in common with those of fungal mitochondrial mRNA and rRNA IVSs, sequences that by genetic analysis are known to be important cis-acting elements for splicing of the mitochondrial RNAs. In the predicted structure of the Tetrahymena IVS, these sequences interact in a pairwise manner similar to that postulated for the mitochondrial IVSs. These findings suggest a common origin of some nuclear and mitochondrial introns and common elements in the mechanism of their splicing.     

Partial amino-acid sequences of human and rabbit C-reactive proteins: homology with immunoglobulins and histocompatibility antigens.

Partial amino-acid sequence analyses of the amino terminus of rabbit C-reactive protein and of a peptide isolated from human C-reactive protein after cyanogen bromide cleavage show an extensive sequence homology between these proteins. Computer analysis detected a distant but significant homology between rabbit C-reactive protein and the CH3 domain of human IgG, In addition, an examination of the limited data available for the amino-acid sequences of human and mouse histocompatibility antigens revealed a similarity between these proteins and C-reactive protein and, therefore, immunoglobulins; These relationships are presented as evidence in support of the hypothesis that C-reactive protein and immunoglobulins share, in addition to functional similarities, a common evolutionary origin with the major histocompatibility antigens.     

Molecular cloning of human epsilon-globin gene.

Human beta-like globin genes were investigated by use of rabbit beta-globin cDNA plasmid as a cross-species hybridization probe. Normal and beta 0/delta beta 0 thalassemic DNA were compared by filter hybridization procedures. It proved possible to demonstrate that the rabbit probe detected G gamma, A gamma, delta, beta, beta 0, and delta beta 0 human globin genes as well as an additional unidentified beta-like globin gene. By use of an agarose gel elution procedure, fractions of HindIII-digested DNA enriched for beta-like globin genes were purified. One of these fractions, 8.0 kilobases in size, was clonedinto lambda 788, and EK2 lambda HindIII vector. A positive clone was obtained and characterized by restriction mapping and sequence analysis. The sequence data obtained predicted an amino acid sequence that exactly matches a part of human epsilon-globin. The human non-alpha-globin locus is now nearly complete. delta, beta, and gamma human globin genes have already been cloned and analyzed. We describe here the cloning of the remaining non-alpha-globin gene, epsilon.     

Adipocyte P2 gene: developmental expression and homology of 5''-flanking sequences among fat cell-specific genes.

We have isolated the mouse gene encoding adipocyte P2, aP2, the differentiation-dependent adipocyte protein homologous to myelin P2. The aP2 gene is present in a single copy in the mouse and is present in single or few copies in species from human to Drosophila. The entire gene spans 4 kilobases and consists of four exons encoding 25, 57, 34, and 16 amino acids; the overall exon structure is similar to the gene encoding liver fatty acid binding protein. A plasmid vector was constructed containing the entire aP2 gene with flanking sequences, modified by linker insertion. When this gene is stably introduced into 3T3-F442A cells, it is expressed only upon adipose differentiation, with a time course of induction very similar to that of the endogenous aP2 gene. We have compared the DNA sequence of the 5'-flanking region of the aP2 gene to the promoter regions of two other genes activated during adipocyte differentiation, glycerol-3-phosphate dehydrogenase and adipsin, and find a 13-base region of homology (Formula: see text) present in multiple copies in the 5'-flanking region of each gene. An adjacent 15-base sequence is present only in glycerol-3-phosphate dehydrogenase and aP2 genes. Both of these elements share homology with putative viral enhancer core sequences. These results indicate that the aP2 gene contains sequence information necessary for differentiation-dependent expression in fat cells; common elements shared by adipocyte-specific genes may play a role in this process.     

Genomic structure of the human caldesmon gene.

The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells. HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLa l-CaD II. The short amino-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h-CaD. We have also identified WI-38 l-CaD I, which contains a 26-amino acid insertion relative to WI-38 l-CaD II. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic structure of the human CaD gene was determined. The human CaD gene is composed of 14 exons and was mapped to a single locus, 7q33-q34. The 26-amino acid insertion is encoded in exon 4 and is specifically spliced in the mRNAs for both h-CaD and l-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaD (residues 73-207 for h-CaD and WI-38 l-CaDs, and residues 68-201 for HeLa l-CaDs). The regulation of h- and l-CaD expression is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change in expression between l-CaD and h-CaD might be caused by this splicing pathway.     

Nucleotide sequences for the gene junctions of human respiratory syncytial virus reveal distinctive features of intergenic structure and gene order.

Complete sequences for the intergenic regions of the genome of human respiratory syncytial virus were obtained by dideoxynucleotide sequencing using synthetic oligonucleotides. These experiments established that the 10 respiratory syncytial viral genes are arranged, without additional intervening genes, in the order 3' 1C-1B-N-P-M-1A-G-F-22K-L 5'. For the first nine genes, the exact gene boundaries were identified by comparison of the genomic sequences with previously determined mRNA sequences. The intergenic regions varied in length from 1 to 52 nucleotides and lacked any obvious conserved features of primary or secondary structure except that each sequence ended (3' to 5') with an adenosine residue. The exact start site of the 10th gene, the L gene, was not determined. However, RNA blot hybridization using a synthetic oligonucleotide designed from the genomic sequence mapped the L gene to within 54 nucleotides of the end of the penultimate 22K gene. The lack of conservation of chain length and nucleotide sequence for the respiratory syncytial viral intergenic regions, together with the complexity of the genetic map, contrasts with previous observations for other nonsegmented negative-strand viruses.     

Molecular evolution of the human adult alpha-globin-like gene region: insertion and deletion of Alu family repeats and non-Alu DNA sequences.

Previous heteroduplex studies have revealed extensive sequence homology between the two human adult alpha-globin-like genes (alpha 2 and alpha 1) and their flanking regions. These homologous regions, which are interrupted by two blocks of nonhomology, each span approximately 4 kilobases [Lauer, J., Shen, C.-K. J. & Maniatis, T. (1980) Cell 20, 119-130]. We have determined 3 kilobases of DNA sequences within and flanking the nonhomologous blocks of these two tandem duplication units. A total of three Alu family repeats has been identified. Two of them are approximately 300 base pairs long and define the 3' ends of the first homology blocks. The third Alu family member is a 600-base-pair-long sequence consisting of two monomeric Alu members arranged in a head-to-tail fashion. It is located in the 3' portion of the first block of nonhomology in alpha 2-gene-containing unit. We present direct evidence that this dimeric Alu sequence was inserted at a staggered break. The second nonhomology block is the result of insertion or deletion of a 224-base-pair sequence. From these data and the calculation of sequence divergence, we propose a history for the evolution of the human adult alpha-globin-like gene region. We also suggest that DNA insertion elements may disrupt gene correction processes in the two duplication units containing alpha 2- and alpha 1-globin genes.     

Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.

Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier.     

Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase.

A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a lambda gt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.     

A human acetylcholinesterase gene identified by homology to the Ace region of Drosophila.

The Ace locus of the Drosophila genome controls biosynthesis of the neurotransmitter-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). We injected the mRNA species hybridizing with DNA fragments from this region into Xenopus oocytes, in which acetylcholinesterase mRNA is translated into active acetylcholinesterase. A 2.0-kilobase (kb) fragment of DNA from this region selectively hybridizes with Drosophila mRNA capable of inducing the biosynthesis of acetylcholinesterase in oocytes. This Drosophila DNA fragment cross-hybridized with human brain poly(A)+ RNA. We therefore used this DNA fragment as a probe for homologous sequence(s) in a human genomic DNA library and thus selected a 13.5-kb human DNA segment. DNA blot-hybridization revealed that a 2.6-kb fragment of this human DNA segment hybridizes with the Drosophila 2.0-kb DNA fragment. Both Drosophila and human fragments hybridized with a human brain mRNA species of about 7.0-kb that was barely detectable in the acetylcholinesterase-deficient HEp carcinoma. A fraction containing mRNA of similar size, extracted from human brain, induced acetylcholinesterase biosynthesis in oocytes. The human DNA fragment also was used in hybridization-selection experiments. In oocytes, hybrid-selected human brain mRNA induced acetylcholinesterase activity that was completely inhibited by 1,5-bis[4-allyldimethylammonium)phenyl]pentan-3-one dibromide but not by tetraisopropyl pyrophosphamide, a differential response to these inhibitors characteristic of "true" human brain acetylcholinesterase. These findings strongly suggest that both the Drosophila and the human DNA fragments are directly involved in controlling acetylcholinesterase biosynthesis.     

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.     

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.     

Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.

Mobile group I introns are hypothesized to have arisen after invasion by endonuclease-encoding open reading frames (ORFs), which mediate their mobility. Consistent with an endonuclease-ORF invasion event, we report similarity between exon junction sequences (the recognition site for the mobility endonuclease) and intron sequences flanking the endonuclease ORF in the sunY gene of phage T4. Furthermore, we have demonstrated the ability of the intron-encoded endonuclease to recognize and cleave these intron sequences when present in fused form in synthetic constructs. These observations and accompanying splicing data are consistent with models in which the invading endonuclease ORF is provided safe haven within a splicing element. In turn the intron is afforded immunity to the endonuclease product, which imparts mobility to the intron.