A 370-kb Cosmid Contig of the Serpin Gene Cluster on Human Chromosome 14q32.1: Molecular Linkage of the Genes Encoding α1-Antichymotrypsin, Protein C Inhibitor, Kallistatin, α1-Antitrypsin, and Corticosteroid-Binding Globulin

The human genes encoding α1-antitrypsin (α1AT, gene symbol PI), corticosteroid-binding globulin (CBG), α1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen–CBG–ATR–α1AT–KAL–PCI–AACT–tel. The genes occupy 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is 12 kb of intergenic DNA between KAL and PCI and 19 kb between PCI and AACT. The proximal subcluster includes α1AT, ATR, and CBG; it occupies 90 kb of genomic DNA, with 12 kb of DNA between α1AT and ATR and 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.     

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.     

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.     

Formation of a large, complex domain of histone hyperacetylation at human 14q32.1 requires the serpin locus control region

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the ~130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1α (HNF-1α) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1α/HNF-4 signaling cascade and the serpin LCR.     

Molecular linkage of the human αl-antitrypsin and corticosteroid-binding globulin genes on Chromosome 14q32.1

The genes encoding α1-antitrypsin (α1AT; gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of structurally related serine protease inhibitor (serpin) genes on human Chromosome (Chr) 14q32.1. This cluster also includes the genes encoding α1-antichymotrypsin (AACT) and protein C inhibitor (PCI), as well as an α1-antitrypsin-related sequence (ATR; gene symbol PIL). In this report we present a detailed restriction map of a 110-kb region of genomic DNA that includes the α1AT, ATR, and CBG genes. Gene order in this interval is tel–α1AT–ATR–CBG–cen, and all three genes are transcribed in a distal-to-proximal orientation. Within the gene cluster, ATR is approximately 12 kb downstream of α1AT, and CBG is about 57 kb downstream of α1AT. Repetitive DNA sequences have been mapped throughout the interval, and several new restriction site polymorphisms in the region are described. Received: 25 May 1997 / Accepted: 23 July 1997     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

A novel porcine gene,alpha-1-antichymotrypsin 2 (SERPINA3-2): sequence,genomic organization,polymorphism and mapping

A novel porcine gene, alpha-1-antichymotrypsin 2 (SERPINA3-2), a member of the serpin superfamily, was isolated from a porcine genomic library and sequenced. The genomic organization of the approximately 9.0 kb gene was determined on the basis of the porcine liver cDNA of SERPINA3-1 and SERPINA3-2, and comprises five exons and four introns. The coding sequence of SERPINA3-2 shares 86% identity with the paralogue, SERPINA3-1. Porcine SERPINA3-2 was found to be an orthologue of human SERPINA3 (71% identity of the coding sequences) and both genes have a similar genomic organization. Polymorphisms were found in intron 4 of the porcine gene using polymerase chain reaction-restriction fragment length polymorphism. The gene was mapped by linkage analysis and radiation hybrid mapping to the distal end of chromosome 7q, to the gene cluster of the protease inhibitors including PI1 (SERPINA1), PI2, PI3, PI4 (apparently paralogues of SERPINA3), and PO1A and PO1B. SERPINA3-2 is the first porcine serpin gene whose genomic organization has been determined.     

Characterization of six new loci within the swine major histocompatibility complex class III region

A search for new potential coding sequences was conducted within two overlapping cosmid genomic DNA clusters of about 170 and 45 kb from the swine major histocompatibility complex class III region. The sequences were detected with various probes, including pools of swine cDNA, homologous and heterologous genomic sequences, and synthetic oligonucleotides. The 170 kb cluster was centered on the tumor necrosis factor genes (TNF), and the 45 kb cluster contained the heat-shock protein 70 genes (HSP70). The TNF cluster revealed the presence of five new genes: lymphotoxin , BAT1, BAT2, BAT3, and a sequence related to DNA-binding factors. No sequence homologous to B144 was found in the TNF cluster, although other unidentified coding sequences may be present in this cluster. The HSP70 cluster contained a gene identified as BAT6, that is, tRNA-valyl synthetase. These results provide new evidence that the genomic maps of these various genes in the TNF and HSP70 sub-regions are similar in swine and human.     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchinStrongylocentrotus purpuratus

Summary The general organization and primary amino acid sequences of theS. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26–27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage.The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.     

Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.     

Nucleotide sequence of the histone gene cluster in the coral acropora formosa (cnidaria; scleractinia): Features of histone gene structure and organization are common to diploblastic and triploblastic metazoans

We report the nucleotide sequence of the core histone gene cluster from the Cnidarian Acropora formosa. This is the first histone gene cluster to be sequenced from a diploblastic organism and the predicted amino acid sequences most resemble those of sea urchin equivalents. Each of the Cnidarian histone genes has two conserved regions 3 of the coding sequences and these closely resemble those of the metazoan a-class histone genes. In A. formosa the core histone genes are arranged as opposed (H3/H4 and H2A/H2B) pairs, a pattern common to the nondeuterostome metazoa, and tandem repetition is the predominant pattern of organization in the Cnidarian. With the recent identification of several classes of homeobox genes in Cnidarians these features clearly align the Cnidaria with triploblastic metazoans, supporting a monophyletic origin of the metazoa.     

Comparative genomics of the keratin-associated protein (KAP) gene clusters in human,chimpanzee, and baboon

We have previously identified a cluster of 16 genes that encode hair-specific proteins, called keratin-associated proteins (KAPs), located on human Chromosome (Chr) 21q22.3. Here, we have identified similar KAP gene clusters in two primates, chimpanzee and baboon. DNA sequence comparison revealed the common cluster structure consisting of 16 KAP genes for these three primates, but a significant difference was found in the baboon. Baboon possesses a new KAP gene not found in human and chimpanzee, whereas one KAP gene (KRTAP18.12) that exists in human and chimpanzee was lost in baboon, making no change in the total number of KAP genes. Interestingly, the sequence for coding regions are highly variable among species owing to insertions and deletions, resulting in variation of gene size. On the contrary, the sequences for the 5 upstream region are highly conserved among species. These findings suggest that the ancestral KAP gene cluster was composed of 17 genes before the divergence of Old World monkeys (baboon) to the anthropoid (human and chimpanzee). The sequence data described in this paper have been submitted to the DDBJ/EMBL/GenBank data library under accession nos. AP006271–AP006274. (Shinsei Minoshima) Present address: Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan.     

Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.     

A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays

The identification of a cDNA (MR19) corresponding to a maize -tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two -tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published -tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3 probes it appears that both genes are preferentially expressed in the radicular system of the plant. The -tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.