Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene

The cytochrome P450 2C (CYP2C) gene locus was found to includea novel exon 1 sequence with high similarity to the canonicalexon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) andPCR amplifications of human liver cDNA revealed the presenceof several intergenic species containing the CYP2C18 exon 1–likesequence spliced to different combinations of exonic and intronicsequences from the CYP2C9 gene. One splice variant was foundto have an open reading frame starting at the canonical translationinitiation codon of the CYP2C18 exon 1–like sequence.Another variant consisted of the nine typical CYP2C9 exons splicedafter the CYP2C18 exon 1–like sequence through a segmentof CYP2C9 5' flanking sequences. Moreover, analysis of bacterialartificial chromosome (BAC) clones revealed that the CYP2C18exon 1–like sequence was located in the intergenic regionbetween the CYP2C19 and CYP2C9 genes. The finding that a solitaryexon is spliced with sequences from a neighboring gene may beinterpreted as representing a general evolutionary mechanismaimed at using the full expression potential of a cell's genomicinformational content.     

Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.     

Unique Telomeric Expression Site of Major-Surface-Glycoprotein Genes of Pneumocystis carinii

Major cell surface glycoproteins (MSG) of Pneumocystis cariniiplay a crucial role in the host-parasite interactions involvedin P. carinii pneumonia in AIDS patients. Genes encoding MSGsare repeated, highly polymorphic, and distributed among allof the 14-15 chromosomes. Here we show, by BAL-31 exonucleasecleavage and DNA cloning experiments, that the unique expressionsite (previously termed UCS) of MSG genes located in the 500-kbchromosome is telomeric. The 11-kb genomic UCS fragment isolatedand sequenced in this study contained one MSG coding sequence(termed msg105), subtelomeric repetitive sequences and telomere-specifictandem repeats of TTAGGG oriented 5' to 3' towards the DNA end.Despite the N-terminal polymorphism, the C-terminal one-thirdsequence of MSG105 was identical to one of the known MSG-cDNAs,suggesting homologous recombination within the MSG coding sequences.These features closely resemble the Variant Surface Glycoproteinsystem of the protozoan parasite Trypanosoma brucei, suggestingthat the genetic heterogeneity of MSGs is generated by recombinationbetween the UCS expression site and multiple MSG genes by meansof reciprocal exchange or gene conversion.     

Identification of a Novel Human Gene Containing the Tetratricopeptide Repeat Domain from the Down Syndrome Region of Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsiblefor the DS main features, has been defined by analysis of DSpatients with partial trisomy 21. Within the DS region, we constructeda 1.6-Mb P1 contig map previously. To isolate gene fragmentsfrom the 1.6-Mb region, we performed direct cDNA library screeningand exon trapping using the P1 clones and a human fetal braincDNA library, and obtained 67 cDNA fragments and 52 possibleexons. Among them, 23 cDNA fragments and 4 exons were interpretedto be derived from a single gene by localization on P1 clonesand by Northern analysis. To obtain the full-length cDNA sequence,longer cDNA clones were further screened from another humancDNA library which was enriched with longer cDNA species. Theseclones were sequenced and assembled to a sequence of 9045 bp.This transcribed sequence encodes a novel 2025 amino-acid proteincontaining tetratricopeptide repeat (TPR) motifs and thereforethe gene was designated as TPRD (a gene containing the TPR motifson the Down syndrome region). The TPR domain has been foundin a certain protein phosphatase and in other proteins involvedin the regulation of RNA synthesis or mitosis. The TPRD gene,the novel gene which was proved to be in the 1.6-Mb region andto have the interesting features described above, is a candidatefor genes responsible for the DS phenotypes.     

Structure and function of bicistronic RNA encoding the phosphoprotein and matrix protein of measles virus.

Two independent full-length replicas of a bicistronic RNA species containing the complete P and M genes of measles virus arranged in tandem were isolated from an expressible cDNA library. Sequences at the 5' and 3' termini suggested that the bicistronic RNA was initiated and terminated at precisely the same locations as the monocistronic mRNAs of the P and M genes, respectively. The P and M cistrons were fused together via an intergenic region which was exactly colinear with and complementary to the intergenic region of the genomic RNA. This RNA species was polyadenylated at the normal polyadenylation site at the 3' terminus of the M cistron, but not in the intergenic region. By DNA-mediated gene transfer, these cDNA clones were expressed into bicistronic RNA containing both P and M sequences in primate cells. RNA thus generated did not undergo nucleolytic processing but was translated into high levels of a 70,000-Mr protein immunoprecipitated by monoclonal antiserum against the measles virus P protein. M protein was not produced in the same cells even though the M cistron could direct M protein synthesis in vitro once excised from the upstream P cistron. These results suggested that bicistronic RNA could direct protein synthesis from the first but not the second cistron and might contribute at least in part to expression of viral genes in vivo.     

Multi-Gene Family of Major Surface Glycoproteins of Pneumocystis carinii: Full-Size cDNA Cloning and Expression

The major surface glycoprotein (MSG) of Pneumocystis cariniiplays a crucial role in the fatal pneumonia caused by this organismin AIDS patients. A cDNA encoding a full-length MSG polypeptidewas isolated from a phage library of rat-derived P. cariniicDNAs. The deduced MSG, referred to as the MSG5 subtype, isa 120,765-Da protein composed of 1,076 amino acids and containsan anchoring hydrophobic sequence at the C-terminus of the protein.Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene familywith 70% protein sequence identity between subtypes. P. cariniikaryotype hybridization analyses indicated that the MSG genefamily members are scattered throughout most of the P. cariniichromosomes. These recombinant MSG proteins reacted with theantiserum from P. carinii-infected rats, as expected, and antiserumgenerated against P. carinii-infected mice, indicating the existenceof common determinants in MSG polypeptides. The family of MSGproteins is rich in cysteine residues and these cysteine arehighly conserved in all MSG subtypes regardless of species specificity,suggesting the structural and/or functional importance of thesecysteine. The pathobiological significance of the MSG gene familyand its sequence diversity in P. carinii is discussed.     

Mouse connexin 45: genomic cloning and exon usage

Connexin 45 is a gap junction protein that is prominent in early embryos and is widely expressed in many mature cell types. To elucidate its gene structure, expression, and regulation, we isolated mouse Cx45 genomic clones. Alignment of the genomic DNA and cDNA sequences revealed the presence of three exons and two introns. The first two exons contained only 5' untranslated sequences, while exon 3 contained the remaining 5' UTR, the entire coding region, and the 3' UTR. An RT-PCR with exon-specific primers was utilized to examine exon usage in F9 mouse embryonal carcinoma cells and adult mouse tissues. In all samples, PCR products amplified using exon 2/exon 3 or exon 3/exon 3 primer pairs were much more abundant than products produced using exon 1/exon 2 or exon 1/exon 3 primer pairs, suggesting that Cx45 mRNAs containing exon 1 were relatively rare compared with mRNAs containing the other exons. Rapid amplification of cDNA ends (5'-RACE) was performed using antisense primers from within exon 3 and template RNA prepared from F9 cells or from adult mouse kidney. We obtained multiple RACE products from both templates, including products that contained all three exons and were spliced identically to the cDNA. However, clones were also isolated (from kidney) that began within the region previously identified as intron 1 and continued upstream with a sequence identical to the cDNA, including splicing to exon 3. These results show that mouse Cx45 has a gene structure that differs from that of previously studied connexins and allows the production of heterogeneous Cx45 mRNAs with differing 5' UTRs. These differences might contribute to regulation of Cx45 protein levels by modulating mRNA stability or translational efficiency.     

Functional characterization of ferret CCL20 and CCR6 and identification of chemotactic inhibitors

CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG_2a fusion protein (fCCL20-mIgG_2a) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.     

Construction and characterization of cDNA clones encoding the 5' end of the chicken pro alpha 1(I) collagen mRNA

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.     

人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。     

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.