Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

The alternative exons 1 of the mouse aromatase cytochrome P-450 gene

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5′ regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5′ regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.     

Organization of the human myoglobin gene.

Cross-hybridization of the grey seal myoglobin gene to human DNA detected a single human myoglobin gene plus an extensive family of sequences apparently related to the central exon of this gene. The functional human gene is 10.4 kb long and has a haemoglobin-like three exon/two intron structure with long non-coding regions similar to its seal homologue. At least 300 bp of 5'-flanking region are closely homologous between the two genes, with the exception of a divergent purine-rich region 68-114 bp upstream of the cap site. A diverged tandem repetitive sequence based on (GGAT)165 is located 1100-1750 bp upstream from the gene; internal homology units within this sequence suggest sequence homogenization by gene microconversions. A second 33-bp tandem repeat element in the first intron is flanked by a 9-bp direct repeat, shares homology with other tandem repetitive elements in the human genome and may represent a novel form of transposable element.     

Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression

We have isolated a chromosomal DNA segment of the human IL-4 gene based on homology with a human IL-4 cDNA sequence and determined its complete nucleotide sequence. The human IL-4 gene, which occurs as a single copy in the haploid genome, is mapped on chromosome 5. It is composed of four exons and three introns and is approximately 10 kilobase pairs in size. 5'-Flanking regions of human and mouse IL-4 genes share about 85% homology extending more than 500 base pairs upstream of a "TATA" like sequence. Several patches of sequences are found in the 5'-flanking region of the human IL-4 gene which are homologous to sequence in the 5'-flanking regions of the IL-2, IL-3, IL-5, and granulocyte-macrophage (GM)-CSF genes. The IL-4 gene is inducible after treatment of human T cell clone by phorbol-12-myristate-13-acetate (TPA) and calcium ionophore A23187. The 2.3-kb 5'-flanking region of the human IL-4 gene transiently transfected into Jurkat human T cell leukemia cells is activated efficiently in response to TPA and A23187 stimulation and, although less efficiently, by human T cell leukemia virus type I-encoded p40x or BPV-encoded E2 protein. Combination of TPA/A23187 and p40x or E2 protein further augmented the level of expression.     

Structure of the human blood platelet membrane glycoprotein Ib alpha gene

The gene for human platelet glycoprotein Ib alpha-chain has been cloned from a genomic cosmid library using a partial cDNA clone as probe. 3530 bp were sequenced including the entire transcribed part, as well as additional 5' and 3' regions. A single intron was found 6 bp upstream of the ATG initiation codon. An exceptionally long exon was identical to the recently published cDNA sequence (1). The 5' upstream promoter region is atypical for eukaryotic genes with only a weak homology to the characteristic promoter consensus sequences. The 3' region contains two repetitive Alu elements, belonging to distinct subfamilies, connected by an oligo(dA) linker.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

Sequence homologies in the protamine gene family of rainbow trout

We have sequenced five different rainbow trout protamine genes plus their flanking regions. The genes are not clustered and do not contain intervening sequences. There is an extremely high degree of sequence conservation in the coding and 3' untranslated regions of the gene. Downstream sequences exhibit little homology though conserved regions are found 250 base pairs 3' to the gene. There are four regions upstream of the gene that are highly conserved in the six clones, including the canonical Goldberg - Hogness box which is 45 base pairs 5' to the coding region. A second homologous region is found 90 bases upstream. Although in the same approximate location as the CAAT box found upstream of other genes, it does not contain the canonical CAAT sequence. Further upstream of the protamine genes at -115 there is an A-T rich sequence while a 25 base pair conserved sequence is located 150 bases upstream. In addition we report the presence of a potential Z-DNA region of predominantly A-C repeats approximately one kilobase downstream of one of the genes.     

Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the δ Subunit of Adaptor-Related Protein Complex AP-3, Not Associated with the Cell Surface

A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (BLVR) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought to be a type I transmembrane protein, the deduced protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding transmembrane and cytoplasmic regions of the predicted bovine BLVR protein. No significant hydrophobic region was found in the mouse protein. Recently, a human cDNA which was highly homologous (69.6% homology) to the mouse BLVR gene was reported. The cDNA encodes the δ subunit of the human adaptor-related protein complex AP-3, which aligned almost collinearly with the mouse BLVR protein. AP-3 and all other related adaptor protein complexes have been shown to be associated with intracellular vesicles but not with the cell surface. Thus, the mouse BLVR homolog appeared to be the mouse AP-3 δ subunit itself or closely related to it, but the bovine BLVR gene seemed slightly different from the adaptor subunit gene family.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

Identification and functional validation of a 5' upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non-coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5' upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at -15 kb. We detected several stretches of homology within the first 30 kb 5' tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5' upstream regulatory sequence found between -8 and -10 kb of the human tyrosinase locus (termed h5'URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at -9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue-specific enhancer in the h5'URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5'URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.