An amplified insect dihydrofolate reductase gene contains a single intron

We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.     

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.     

Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element.

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.     

Deoxyribonuclease I sensitivity of the ovomucoid-ovoinhibitor gene complex in oviduct nuclei and relative location of CR1 repetitive sequences

The location of CR1 middle repetitive sequences within or near the boundaries of the ovalbumin DNase I sensitive domain has suggested that CR1 sequences may play a role in defining transition regions of DNase I sensitivity in hen oviduct nuclei. We have examined this apparent relationship of CR1 sequences and transitions of chromatin structure by determining the DNase I sensitivity in oviduct nuclei of a 47-kilobase region that contains five CR1 sequences and the transcribed ovomucoid and ovoinhibitor genes. We find that three of the CR1 sequences occur within a broad transition region of decreasing DNase I sensitivity downstream of the ovomucoid gene. Another CR1 is in a region of decreased DNase I sensitivity within the ovoinhibitor gene. The fifth CR1 sequence is in a DNase I sensitive region between the two genes but which is less sensitive to DNase I digestion than the region immediately upstream from the ovomucoid gene. Thus, the CR1 sequences occur within regions of reduced relative DNase I sensitivity, suggesting that CR1s could facilitate the formation of a chromatin conformation that is less sensitive to DNase I digestion. Unexpectedly, the noncoding strand of sequences within and immediately adjacent to the 5' end of the actively transcribed ovomucoid and ovalbumin genes was less sensitive to DNase I digestion than their respective coding strands.     

DNase I hypersensitive sites and transcriptional activation of the lamin A/C gene.

The lamin A/C gene encodes subtypes of nuclear lamins, which are involved in nuclear envelope formation, and was recently identified as the responsible gene for the autosomal dominant Emery-Dreifuss muscular dystrophy. Expression of the lamin A/C gene is developmentally regulated but little is known about the regulatory mechanism. Previous studies of lamin A/C expression suggested that the chromatin structure is important for the regulation of its expression. To elucidate the regulatory mechanism of the lamin A/C gene expression, we have analysed the functional region of the mouse lamin A/C promoter and the chromatin structure of the gene in terms of nucleosome structure and DNase I hypersensitivity. Our analyses revealed disruption of the nucleosome array at the promoter region and the presence of multiple DNase I hypersensitive sites (HSs) which were specifically associated with expression of the lamin A/C gene. Inclusion of a segment which contained the HSs in a lamin A/C promoter-luciferase reporter plasmid showed no effect on the transfected promoter activity in transient expression assays. On the other hand, substantial enhancement of the promoter activity was detected when the transfected DNA was stably integrated into the genome, suggesting the importance of the HSs in the regulation of lamin A/C expression.     

Analysis of DNase 1 sensitivity and methylation of active and inactive X chromosomes of kangaroos (Macropus robustus) by in situ nick translation

The overall nuclease sensitivity and methylation of active and inactive X chromosomes of kangaroos were examined by in situ nick translation. Cultured fibroblasts of subspecies wallaroo-euro (Macropus robustus robustus; Macropus robustus erubescens) hybrids were used, enabling the paternally and maternally derived X chromosomes to be distinguished. No difference was found between the active and inactive X chromosomes with DNase I or MspI digestion. When chromosomes were digested with the methylation sensitive restriction enzymes HpaII and HhaI, the inactive X chromosome was labelled to a greater extent. These results indicate no overall difference in chromatin condensation between the active and inactive X chromosomes and greater overall methylation of the active X chromosome. This relative undermethylation of the inactive X chromosome may be important in X chromosome inactivation, but its function, if any, remains to be determined.by A. Bird