Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.

Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by the monoclonal antibodies HMFG-1, HMFG-2, and SM-3. Previously isolated partial cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 20-amino acid repeat units. We now report the full sequence for PEM, as deduced from cDNA sequences. The encoded protein consists of three distinct regions: the amino terminus consisting of a putative signal peptide and degenerate repeats; the major portion of the protein which is the tandem repeat region; the carboxyl terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail. Potential O-glycosylation sites (serines or threonines) make up more than one-fourth of the amino acids. Length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus. Tandem repeats appear to be a general characteristic of mucin core proteins.     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

Characterization of a highly polymorphic region 5′ to JH in the human immunoglobulin heavy chain

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.     

Organization of the gene for gelatin-binding protein (GBP28)

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.     

Cloning and chromosome mapping of the human interleukin-1 receptor antagonist gene.

By screening a human genomic library with an interleukin-1 receptor antagonist (IL-1ra) cDNA probe, we have isolated a 15 kb clone which contains the entire coding region of the gene as expressed in monocytes, and includes 6 kb of 5'-upstream sequence. The gene contains four exons which code for the secreted form of the IL-1ra, however, our clone does not contain the alternative first exon used to generate an intracellular form of the protein as the protein as found in epithelial cells. Analysis of the sequence reveals a consensus TATA box, and three Alu repeats, two of which are in the upstream region and one in intron 3. The sequence also reveals an 86 bp motif tandomly repeated four times within intron 2, and may reflect the polymorphism known to exist in this region of the gene. By in-situ fluorescence hybridization we have shown that the IL-1ra gene is found on the long arm of chromosome 2 and maps to 2q13-14.1. Previous studies have revealed that IL-1 alpha, and IL-1 beta and both type I and type II forms of the IL-1 receptor all map close to this region of chromosome 2.     

TRbase: a database relating tandem repeats to disease genes for the human genome

MOTIVATION: Tandem repeats are associated with disease genes, play an important role in evolution and are important in genomic organization and function. Although much research has been done on short perfect patterns of repeats, there has been less focus on imperfect repeats. Thus, there is an acute need for a tandem repeats database that provides reliable and up to date information on both perfect and imperfect tandem repeats in the human genome and relates these to disease genes. RESULTS: This paper presents a web-accessible relational tandem repeats database that relates tandem repeats to gene locations and disease genes of the human genome. In contrast to other available databases, this database identifies both perfect and imperfect repeats of 1-2000 bp unit lengths. The utility of this database has been illustrated by analysing these repeats for their distribution and frequencies across chromosomes and genomic locations and between protein-coding and non-coding regions. The applicability of this database to identify diseases associated with previously uncharacterized tandem repeats is demonstrated.     

Detection of novel genetic markers by mismatch analysis.

Chemical mismatch detection has been used to identify previously unknown genomic sequence variations that represent a new source of markers for genetic analysis. The approach detects all types of sequence changes, and therefore overcomes the limitation of restriction analysis, which identifies only a small fraction of the available sequence variations. Three new markers identified at the 3' end of the human dystrophin gene result from variable numbers of exact tandem repeats of 4bp (two examples) or 5bp (one example). None of these would have been detected as restriction fragment length polymorphisms by established procedures.     

Genomic organization of human 5 S rDNA and sequence of one tandem repeat

An organization of human 5 S rDNA repeats is inferred from Southern analyses of restriction digests of genomic DNA fractionated by pulsed-field and conventional gel electrophoreses. A single unit of 2.2 kb is repeated approximately 90 times within a 200-kb fragment (defined by enzymes that do not cleave within individual units, i.e., EcoR1, BglII, HindIII, and PvuII); a comparable number of 5 S sequences are scattered elsewhere in the genome. A lambda clone containing six complete 5 S repeats was obtained from a human placental DNA library. One repeat contains 2231 bp and includes poly(dG-dT).(dC-dA), tracts of polypyrimidine, and an Alu sequence in the spacer region. Also, 5-S-hybridizing clones, containing DNA inserts with an average size of 250 kb, have been obtained as yeast artificial chromosomes. Thus far, four clones have been partially characterized and shown to be 5 S sequences from loci separate from the tandem repeat units.     

A second gene in a Balbiani ring. Chironomus salivary glands contain a 6.5-kb poly(A)+ RNA that is transcribed from a hierarchy of tandem repeated sequences in Balbiani ring 1

A second gene has been discovered at a previously studied Balbiani ring in Chironomus. Northern hybridizations demonstrated that cDNA clone pCt35 originated from a salivary gland specific 6.5-kilobase (kb) RNA that was abundant, nonribosomal, and apparently poly(A)+. pCt35 had a 120-base pair (bp) insert with 1.6 copies of a 75-bp sequence that contained two open reading frames. Southern hybridizations indicated that pCt35 was homologous to at least a 4-kb block of genomic DNA organized as a hierarchy of 150- and 300-bp tandem repeats. In situ hybridization localized these sequences to Balbiani ring 1. From these results we postulated that a 6.5-kb RNA gene may have evolved by stepwise duplication and amplification of a 75-bp ancestral sequence.     

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.     

Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases

The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus. Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs. A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli. Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins. Analysis of PCR fragments amplified from the genomes of N. tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N. tabacum gene. These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

Identification, mapping, and genomic structure of a novel X-chromosomal human gene (SMPX) encoding a small muscular protein

Reciprocal probing has been used to identify a cDNA clone (xh8H11) representing a gene preferentially expressed in striated muscle. The gene maps close to DXS7101 31.9 cM from the short arm telomere of the X-chromosome at Xp22.1. On searching expressed and genomic databases, 21 expressed sequence tags were found that allowed the assignment of a human extended consensus sequence of 887 bp, suggesting a completely expressed gene symbolized as SMPX. By using the human consensus sequence, the orthologous mouse Smpx and rat SMPX genes could be aligned and confirmed by complete sequencing of additional SMPX-related clones obtained by library screening. An open reading frame was identified encoding a peptide of 88-86 and 85 amino acids in human and rodents, respectively. The predicted peptide had no significant homologies to known structural elements. The human consensus cDNA sequence was used to define the genomic structure of the human SMPX that had been missed by a previous large scale sequencing approach. The gene consists of five exons (> or =172, 57, 84, 148, > or =422 bp) and four introns (3639, 10410, 6052, 31134 bp) comprising together 52.1 kb and is preferentially and abundantly expressed in heart and skeletal muscle. Thus, a novel human gene encoding a small muscular protein that maps to Xp22.1 (SMPX) has been identified and structurally characterized as a basis for further functional analysis.