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.     

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.     

Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus

The gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promotor sequence (TTTTCC for the -35 region and TATTGT for the -10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upstream of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34,266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.     

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5′-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.     

Genomic organization of the mouse OSF-1 gene.

The mouse OSF-1 protein (also known as pleiotrophin, HB-GAM, HBGF-8, or HBNF) gene was isolated from a mouse genomic library and sequenced. OSF-1 is a 15-kD secreted protein specifically expressed in bone and brain, and is believed to play a role in brain development and osteogenesis. The mouse OSF-1 gene consists of at least 5 exons and 4 introns and spans > 32 kb. Computer analysis of approximately 4 kb of 5'-flanking sequence of the OSF-1 gene revealed two candidate promoter regions. One candidate promoter contains a thyroid hormone/retinoic acid-responsive element and the other contains two glucocorticoid-responsive elements. DNA sequence analysis of novel OSF-1 cDNA clones indicates that two promoters can be utilized in MC3T3-E1 osteoblastic cells. The overall organization of the mouse OSF-1 gene is similar and the locations of the three exon-intron junctions within the coding region are identical to the mouse gene encoding the differentiation-related factor midkine (MK). Based on this similarity and on the high degree of nucleotide sequence homology (approximately 55%) of mouse OSF-1 and mouse MK, we conclude that OSF-1 and MK are generated from a common ancestral gene and are members of a family of structurally and probably functionally related proteins.     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

Characterization of the beta2-microglobulin gene of the horse

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.     

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.     

Isolation and characterization of non-muscle tropomyosin cDNAs of human and mouse origin

Several cDNA clones of human and mouse non-muscle tropomyosin have been isolated. All the human clones possess a common 23 bp sequence immediate 5' of the initiation codon. However, in the further upstream regions, the nucleotide sequences diverge. Two of the mouse cDNA clones pPSI-8 and pPSI-14 have identical nucleotide sequence in the coding region sequenced. However, 5' of the initiation codon these clones have only 40 identical nucleotides and further upstream the nucleotide sequences diverge. Analysis of the genomic DNAs of mouse cells indicated the possibility of a common gene giving rise to both the tropomyosin cDNAs differing in their 5' ends.