Cloning of genomic sequences of human prointerleukin 1 beta

The human brain library carried in the EMBL3 vector was employed for isolating prointerleukin 1 beta genomic sequences using three synthetic 20-member oligonucleotides. Oligonucleotides were homologous to the following mRNA regions: 3'-nontranslated region/C1/, 3'-translated region of mRNA/C2/ and the sequence coding N-terminal of mature protein/N1/. The oligonucleotide labeling utilized the terminal nucleotidyltransferase and [alpha-32P] dATP and specific activity of labeled oligonucleotides reached 1.6.10(10) cpm. The sizes of the synthesized labeled sequences (tails) were about 10 b.p. Hybridization probe C1 was used for the first screening and 24 hybridization positive clones were detected. For the next screening probe C2 was used and only 2 hybridization positive clones with different level of hybridization were detected from 24 clones. Probe N1 was used for the third screening and allowed to identify the only positive clone. The characterization by restriction mapping and Southern blot analyses have shown that recombinant phage DNA contains all three exons, coding the mature interleukin 1 beta. Some fragments were recloned to tg130 vector phage and nucleotide sequences of exon 5 (completely) and exon 7, intron 4 and 5 (partially) were determined.     

The murine interleukin 1 beta gene: structure and evolution.

We have isolated from a genomic library a murine recombinant clone containing the gene coding for interleukin-1 beta m-RNA. A 7000 b.p. DNA fragment has been sequenced. Sequences homologous with human IL-1 beta cDNA have been found distributed within 7 exons. The translation of these sequences allows the prediction of a protein 269 aminoacids long. Hybridization of P388D1 RNA from cells stimulated with phorbol myristic acetate with a genomic DNA probe shows the existence of a 1.6 Kb murine IL-1 beta mRNA which is absent in the unstimulated cells. The comparative analysis between the murine IL-1 beta and the human IL-1 alpha genes shows extreme conservation of the aminoacids at the exon junctions. This observation together with the similarity in number and size of the exons suggests that these genes have diverged from a common ancestor.     

The Structure of the Gene Encoding the Human Skeletal Muscle α1Subunit of the Dihydropyridine-Sensitive L-type Calcium Channel (CACNL1A3)

The structure of the gene encoding the human skeletal muscle α₁subunit (CACNL1A3) of the dihydropyridine-sensitive voltage-dependent calcium channel was determined by isolation of overlapping genomic DNA clones from human cosmid, phage, and P1 libraries. Genomic fragments containing exons were subcloned, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the CACNL1A3 gene, which spans 90 kb and consists of 44 exons, will facilitate the search for additional mutations in CACNL1A3 that cause neuromuscular disease.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.     

A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Specific isolation of 3'-terminal exons of human genes by exon trapping.

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.     

Human pregnancy-specific beta 1 glycoprotein is encoded by multiple genes localized on two chromosomes.

A human genomic library was screened with a mixture of two cDNA probes, with one covering the 5' coding sequence and the other containing the 3'-end portion of human pregnancy-specific beta 1 glycoprotein (SP1). Seventeen clones were identified, all of which carried insert fragments capable of hybridizing with the cDNA probe. Insert size of these clones varied from 15.0 to 19.8 kb. Partial restriction maps were constructed, which demonstrated the presence of at least seven groups of unique SP1 genomic clones and suggested the possibility of multiple genes coding for SP1. The multigene nature of SP1 was confirmed by hybridization of the SP1 cDNA probe to multiple bands on Southern blots of human genomic DNA. Further analysis with chromosomal DNA dot blot demonstrated the presence of homologous sequences on the X chromosome and autosomal chromosome 6. Thus, human SP1 is apparently coded for by more than one gene residing on the X and 6 chromosomes.     

Construction of a genome library from a beta-0-thalassemic individual from Ferrara: characterization of clones containing beta globin genes

A library of genomic DNA was prepared from a patient with beta o Ferrara thalassaemia: random human DNA fragments (15 - 20 Kb) have been joined to phage lambda vectors and cloned has viable phage particles (4). 4x10(5) phages have been screened for their content in beta globin gene sequences, using a human beta cDNA plasmid (5) as hybridization probe. Five positive clones have been isolated and characterized by restriction endonuclease cleavage analysis and by the hybridization experiments. The results obtained allow the precise localization of the human fragments inside the beta like globin gene cluster (6). The comparison of the thalassaemic fragments with the normal DNA (6 - 7) shows two different restriction endonuclease sites, for Xba I and Eco RI respectively, downstream from the human beta globin gene.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.