John O. Bishop

Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect.

Unusual aberrant expression of a foreign gene in a particular transgenic mouse line is often attributed to chromosomal position effect, although proof of this is lacking. An alternative explanation is that expression has been modified by the arrangement of multiple copies of the foreign gene at the insertion site or by mutation or gene rearrangement. We have distinguished between these explanations in the case of one particular transgenic line by recovering the aberrantly expressed foreign DNA and reintroducing it into the mouse genome to produce secondary transgenic mice. The expression pattern of the gene in the secondary transgenic mice was normal, showing that this case of aberrant expression is due to a chromosomal position effect.

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three of four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.

A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells

Abstract A method is described for sedimenting RNA on a preparative scale in the presence of 85% formamide. It is likely that the RNA is fully denatured, since under the same conditions ribosomal RNA develops full hyperchromicity, and formaldehyde-treated and untreated ribosomal and nuclear RNA sediment in an identical manner. RNA from immature duck red blood cells was fractionated on sucrose gradients. The amount of hemoglobin mRNA and poly(A) in each fraction was measured by hybridization with complementary DNA and poly(U), respectively. We observed that hemoglobin mRNA forms aggregates in the presence of phenol, which are dispersed on formamide gradients. Nuclear RNA became more heavily aggregated, and again the aggregates were dispersed by formamide. A possible nuclear precursor of hemoglobin mRNA was identified. The molecular weight of the precursor is 6–7 × 10 5 , three times as long as hemoglobin mRNA, and it is attached covalently to a poly(A) sequence.

Effects of Thyroid Hormone on Embryonic Oligodendrocyte Precursor Cell Developmentin Vivoandin Vitro

Abstract The oligodendrocyte precursor cell divides a limited number of times before terminal differentiation. The timing of differentiation depends on both intracellular mechanisms and extracellular signals, including mitogens that stimulate proliferation and signals such as thyroid hormone (TH) and retinoic acid (RA) that help trigger the cells to stop dividing and differentiate. We show here that, both in vivo and in vitro, TH is required for the normal development of rodent optic nerve oligodendrocytes, although in its absence some oligodendrocyte development still occurs, perhaps promoted by signals from axons. We also demonstrate that TH from both mother and pup plays a part in oligodendrocyte development in vivo. Finally, we show that precursors in embryonic nerve cultures differ from those in postnatal cultures in two ways: they respond much better to TH than to RA, and they respond more slowly to TH, suggesting that oligodendrocyte precursor cells mature during their early development.

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Abstract The annealing of ribosomal, hnRNA and messenger RNA to DNA in vast DNA excess was examined for material derived from two mammalian (human and rat) and one insect (mosquito) species. Both the haploid DNA content and the kinetic complexity of mosquito DNA are three times smaller than for mammals. However, the annealing pattern of mosquito DNA closely resembles that of mammals: in both cases about 10% of the DNA sequences are rapidly renaturing, 10–15% are of intermediate repetitiousness, and the remainder are essentially unique. Ribosomal RNA hybridizes with a single transition corresponding to 200 genes per haploid chromosome complement in the rat and 430 in the mosquito. Mosquito and mammalian hnRNA are similar, each showing two principal transitions: 30% complementary to intermediate repetitious, and the remainder to unique sequences. The properties of poly(A)-containing mRNA differ appreciably. Mosquito mRNA hybridizes mainly to unique sequences, although there appears to be a minor transition amounting to less than 10% of the total hybridized RNA. In contrast, the mRNA from both mammals contains an appreciable amount of material of intermediate repetitiousness which amounts to 30% of the total hybridization transition.

A study of foldback DNA.

Nuclear DNA from eucaryotes contains a significant fraction which forms duplexes very rapidly and also independently of the DNA concentration. This fraction can be isolated by adsorption to hydroxylapatite and has been called foldback DNA (Britten and Smith, 1970). Here we extend previous studies to show that the foldback fraction is due to the existence of a finite number of foldback foci in each genome equivalent of DNA, approximately 10(5) in the case of Xenopus laevis. More significantly, we have isolated the foldback fraction in quantity from DNA of such a size (in one case broken randomly and in another digested with a restriction endonuclease) that only about 10% of the total DNA has foldback properties. If the foldback foci were located in precisely the same positions in all sets of the Xenopus laevis genome, the prediction would be that these foldback fractions would contain sequences representing 20% (random shear) and 10% (restriction endonuclease) of the total genome. In contrast, our results show that in both cases the foldback fraction contains the entire Xenopus laevis DNA sequence. One possible explanation of these observations is that as in procaryotes, eucaryotic DNA is randomly cross-linked. We show that cross-linkage of Xenopus laevis DNA is not sufficient to explain our observations. In consequence, we have adopted the hypothesis that the formation of foldback DNA is mainly an intrastrand phenomenon, but nevertheless occurs at different sites in different sets of the Xenopus laevis genome.

Initiation of haemoglobin polypeptide chains

Abstract 1. 1. Ribosome fractions, prepared by differential centrifugation of a lysate of rabbit reticulocytes, contained different proportions of polyribosomes, ribosomal monomers (monosomes) and native subunits. In the cell-free system polyribosomes of all fractions synthesised on average one half polypeptide chain per constituent monomer. 2. 2. Chain synthesis de novo also occurred in the cell-free system, to a greater extent in the lighter of the fractions prepared by differential centrifugation. Since large quantities of monosomes were present in all fractions it is unlikely that monosomes are responsible for the initiation of polypeptide chains. An alternative hypothesis, that chain initiation requires the participation of native ribosomal subunits, is supported by several lines of evidence. 3. 3. The heaviest ribosome fraction, containing about 50 % polyribosomes but only 5 % subunits, had little capacity to initiate polypeptide chains. Chain initiation was increased by adding small amounts of a fraction rich in subunits. Zone sedimentation showed the stimulatory activity to sediment at about 50 S. 4. 4. Amongst a number of preparations of the ribosome fractions, a linear relationship could be demonstrated between chain initiation and subunit content, over a limited range. 5. 5. During incubation in the cell-free system the amount of ribosomal subunits declined. A parallel fall occurred in the capacity of the ribosome population to initiate polypeptide chains.

Messenger RNAs coding for mouse major urinary proteins are differentially induced by testosterone

We have investigated the sexual dimorphism of the mouse major urinary proteins (MUPs) by isoelectric focusing (IEF). In each of two inbred strains which have different male patterns (C57BL and BALB/c), the females show a simpler pattern with fewer prominent components. The main female component is different in each strain, and these may be the products of allelic structural genes. Treatment with testosterone induces the excretion of MUPs with the male pattern. The in vitro translation of hybrid-selected MUP mRNA was studied in the same way. Again, the female patterns were simpler than the male, and the patterns obtained with female mRNA from the two inbred strains were different. When the females were treated with testosterone, a male or male-like pattern was obtained. We argue that some MUP structural genes are quite actively transcribed in females and that the transcription of others is dependent to different degrees upon induction by testosterone.

Variation in mouse major urinary protein (MUP) genes and the MUP gene products within and between inbred lines

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.

An analysis of cytoplasmic RNA populations in Drosophila melanogaster, Oregon R

Nucleic acid reassociation methods were used to estimate the number of different polyadenylated RNA [poly (A) + RNA] sequences in the cytoplasm of whole Drosophila melanogaster at different stages of development and in the cytoplasm of cells of the L3 cell line. The number-average length (LN) of poly (A) + RNA from L3 cells is 1.4 kb, and the steady-state LN of the poly (A) tracts is 70 nucleotides. Analysis of RNA-driven reassociation with copy DNA shows that the poly (A) + RNA from L3 cells contains 5200 different sequences distributed in three abundance classes. The RNA forms hybrid duplexes with about 4.5% of single-copy Drosophila DNA, corresponding to 6500 sequences of 1.4 kb. The LN of poly (A) tracts present in whole embryos, larvae, pupae, and imagos is in each case close to 70 nucleotides. RNA-driven reassociation experiments show that poly (A) + RNA from these sources contains, respectively, 3500,≥4900, 6900, and ≥4900 sequences. Cross-hybridization reactions show extensive homology between these RNA populations. All five poly (A) + RNA preparations contain a prominent component with a sedimentation coefficient of 13 and a size of 1.78 kb, which is shown to be the larger species of mitochondrial rRNA. This rRNA binds to oligo (dT) cellulose and serves as an efficient template for cDNA synthesis by reverse transcriptase. In RNA-driven reassociation experiments it behaves like an abundant mRNA.