Characterization of the circadian system of NGFI-A and NGFI-A/NGFI-B deficient mice.

The genes NGFI-A (also known as EGR-1, zif/268, and Krox-24) and NGFI-B (nur/77) have previously been shown to be induced in the SCN of rats and hamsters by photic stimulation during the subjective night. The purpose of this study is to determine whether these genes are also induced in the SCN of mice and, if so, to characterize the circadian system of animals in which either NGFI-A or both NGFI-A and NGFI-B were eliminated by homologous recombination. In wildtype mice, NGFI-A mRNA was found to be induced in the SCN as in other rodent species. Therefore, wheel-running activity was recorded from null mutants and wildtype controls under LD 12:12 and DD conditions. Mice of all three strains appeared to entrain normally to LD 12:12 and could re-entrain to both phase advances and phase delays of the light cycle. The response of the circadian pacemaker of all three genotypes to acute light pulses appeared to be normal. The retinal innervation of the SCN in NGFI-A-/- mice and the photic induction of Fos in the SCN of both NGFI-A-/- and NGFI-A-/-/B-/- mice were indistinguishable from wildtype mice. These results indicate that induction of NGFI-A and NGFI-B is not required for photic entrainment or phase shifting of the mouse circadian system.

Differential expression of insulin-like growth factor genes in rat central nervous system.

A sensitive solution-hybridization assay was used to investigate the expression of genes encoding insulin-like growth factors I and II (IGF-I and -II) in the rat central nervous system (CNS). mRNAs for both IGFs are synthesized throughout the CNS of adult rats but exhibit distinct regional differences for each growth factor. IGF-I mRNA is 8-10 times more abundant in the cervical-thoracic spinal cord and in the olfactory bulb than in whole brain and is enriched 3-fold in the midbrain and cerebellum. IGF-II mRNA is minimally enriched in the medulla-pons and cerebellum but is 3-5 times less abundant in the midbrain and corpus striatum than in total brain. During CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines by a factor of 3-4 at birth, to values found in adult brain. Embryonic neurons and glia synthesize IGF-I mRNA during short-term cell culture; only glia produce IGF-II mRNA. These observations show that IGF-I and IGF-II are differentially expressed in the developing and adult CNS and suggest that each growth factor may play a unique role in the mammalian nervous system.

Insulin-like growth factor gene expression during rat embryonic development.

The function of insulin-like growth factors I and II (IGF-I and IGF-II) in embryogenesis is unknown. To investigate the ontogeny of IGF gene expression during mammalian development we used a highly sensitive and specific solution-hybridization assay to determine the steady state levels of IGF mRNAs during midgestation in the rat. IGF-I mRNA can be detected as early as day 11 of embryonic development and rises 8.6-fold over the ensuing 48 h. By contrast IGF-II mRNA is relatively constant over days 11-14 of gestation. These observations suggest that both IGFs may play important roles in early fetal development.

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.

Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line.

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.

Cloning of the initiation region of a mammalian chromosomal replicon.

In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic and biochemical evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. We have attempted to establish a system in which functional chromosomal origins could be identified in vivo before their isolation by molecular cloning. For this purpose, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence, and which includes the gene for dihydrofolate reductase (DHFR). We have shown that initiation of DNA synthesis within each repeated unit (amplicon) is restricted to a small subset of restriction fragments at the beginning of the S period, suggesting that these fragments contain or flank origins of DNA synthesis. Here we report molecular cloning and restriction mapping experiments showing that all of these early-labelled fragments (ELFs) are derived from a single locus within each repeated unit. This result implies that synthesis of each amplicon initiates from a single origin of replication at the onset of S, and that an amplicon is formally equivalent to a replicon.

Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.