Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes

The complete human dihydrofolate reductase (DHFR) gene has been cloned from four recombinant lambda libraries constructed with the DNA from a methotrexate-resistant human cell line with amplified DHFR genes. The detailed organization of the gene has been determined by restriction mapping of the cloned fragments and DNA sequencing of all the protein coding regions and adjacent intron segments, and shown to correspond to that of the native human DHFR gene. The gene spans a length of approximately 29 X 10(3) bases from the ATG initiator codon to the end of the 3' untranslated region, and contains five introns that interrupt the protein coding sequence. The number and positions of introns are identical to those found in the mouse gene. By contrast, the size of the homologous introns (with the exception of the first one) varies greatly, up to several fold, in the genes from man, mouse and Chinese hamster; the intron sequences also exhibit a great divergence, except in the junction regions. A striking sequence homology, extending over several hundred nucleotides, exists between the human and mouse gene 5' non-coding regions. These regions are characterized by an unusually high G + C content, 72% and 66% in the human and mouse genes, respectively, which is maintained in the first coding segment and first intron, and is in sharp contrast to the relatively low G + C content (approximately 40%) of the remainder of the gene.     

Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development

A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined. The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb. Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons. There are eight introns in both the human and the hamster des genes. Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved. A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns. These sequences all belong to the human AluI family. When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al. [Cell 43 (1985) 327-338] for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes [Minty and Kedes, Mol. Cell. Biol. 6 (1986) 2125-2136] in the 5'-flanking region. The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene. Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.     

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.     

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.     

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20 q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3′ end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3′ end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitiative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5′ flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.     

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.     

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.     

Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.     

Comparative analysis of the structural organization of two closely related vitellogenin genes in X. laevis

The structural organization of the two closely related vitellogenin genes A1 and A2 has been determined and compared by electron microscopy. In both genes the mRNA-coding sequence of 6 kb is interrupted 33 times, leading to a total gene length of 21 kb for gene A1 and 16 kb for gene A2. Thus both genes have a mean exon length of 0.175 kb, while the mean intron length is 0.45 kb in gene A1 and 0.31 kb in gene A2. Because the introns interrupt the structural sequence at homologous positions in genes A1 and A2, we suggest that these two genes are the products of a duplication of an ancestral gene which had an intron-exon arrangement similar to that of the extant genes. Since the duplication event, the sequence and length of the analogous introns have changed rapidly, whereas homologous exons have diverged to an extent of only 5% of their sequences. The results suggest different mechanisms of evolution for exons and introns. While the exons evolved primarily by point mutations, such mutations, as well as deletion, insertion and duplication events, were important in the evolution of the introns.     

The chicken carbonic anhydrase II gene: evidence for a recent shift in intron position.

The complete nucleotide sequence of the coding region of the chicken carbonic anhydrase II (CA II) gene has been determined from clones isolated from a chicken genomic library. The sequence of a nearly full length chicken CA II cDNA clone has also been obtained. The gene is approximately 17 kilobase pairs (kb) in size and codes for a protein that is comprised of 259 amino acid residues. The 5' flanking region contains consensus sequences commonly associated with eucaryotic genes transcribed by RNA polymerase II. Six introns ranging in size from 0.3 to 10.2 kb interrupt the gene. The number of introns as well as five of the six intron locations are conserved between the chicken and mouse CA II genes. The site of the fourth intron is shifted by 14 base pairs further 3' in the chicken and thus falls between codons 147 and 148 rather than within codon 143 as in the mouse gene. Measurements of CA II RNA levels in various cell types suggest that CA II RNA increases in parallel with globin RNA during erythropoiesis and exists only at low levels, if at all, in non-erythroid cells.     

Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family.

We report the complete sequence of the gene encoding mouse glial fibrillary acidic protein (GFAP), the intermediate filament (IF) protein specific to astrocytes. The 9.8 kb gene includes nine exons separated by introns ranging in size from 0.2 to 2.5 kb. A comparison of the organization of the GFAP gene with that of genes encoding other IF proteins reveals that the structure of IF genes is highly conserved in spite of considerable divergence at the amino acid level. Thus, most of the evolutionary events leading to the placement of introns in IF genes must have occurred prior to the duplication and subsequent divergence of IF genes from a presumptive common ancestral sequence. The conserved gene organization is unrelated to structural features of IF proteins. A curious feature of the GFAP gene is the large number of repeated sequences found in the introns. Six tracts of reiterated di- or trinucleotides are present, plus tandem repeats of two different novel sequences. One repeat is unique to the GFAP gene; the other occurs elsewhere in the mouse genome, although at relatively low frequency.     

Construction and expression of mouse thymidylate synthase minigenes

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.     

Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.     

Hamster cells with increased rates of DNA amplification, a new phenotype

Baby hamster kidney (BHK) cells selected simultaneously with N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) gave rise to doubly resistant colonies at frequencies 20 to 260 times greater than the product of the independent frequencies found with PALA or MTX alone. Double resistance was due to amplification of both target genes, CAD and DHFR. Four independent doubly resistant "MP" lines were selected and characterized. Cells resistant to coformycin, pyrazofurin, or ouabain were generated from all four MP lines at rates up to 25 times greater than the rates for BHK cells. These three drugs select cells that have amplified the genes for their target enzymes. Therefore, we conclude that the four MP lines have an amplificator phenotype. All four grew much more slowly than BHK cells, indicating that the amplificator phenotype may be linked to significant defects in metabolism or cell division.     

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.