Purification of globin messenger RNA from dimethylsulfoxide-induced friend cells and detection of a putative globin messenger RNA precursor

Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate ³²P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. ³²P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified ³²P-labeled globin mRNA by RNAase T₁ digestion. The partial sequences for nine oligonucleotides corresponded to those predicted from the amino acid sequences of α and β globin; the other oligonucleotides were presumably derived from non-translated regions.In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [³²P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S.     

beta-Like globin RNA sequences in hemoglobin Lepore disease.

A Southern Italian patient homozygous for hemoglobin Lepore disease synthesizes approximately 3% Lepore delta beta-globin chains (relative to alpha chains) in the reticulocytes. Measurement of beta-like RNA sequences by hybridization to complementary DNA specific for beta-globin demonstrates a low level (1--2% relative to alpha sequences) of these sequences in cytoplasmic RNA from reticulocytes or spleen cells, suggesting that the Lepore gene is expressed into mRNA at a lower extent than normal alpha or beta genes; the comparison with the level of beta-like sequences found in nuclear RNA (6--8%) further supports this conclusion and indicates, in addition, that Lepore RNA might be degraded at a faster rate than normal. 2--3% beta-like sequences are found in nuclear RNA in three cases of homozygous beta0-thalassemia, setting the highest possible estimate for the delta-RNA level; this figure suggests that the 'delta-promoter'-dependent Lepore delta beta gene is somehow more actively expressed than the delta gene.     

Size heterogeneity of polyadenylate sequences in mouse globin messenger RNA

Heterogeneity in the length of the poly(A) region has been demonstrated in mouse α and β-globin messenger RNAs. This finding is based on the initial observation that only 30% of the globin mRNA purified by oligo(dT)-cellulose affinity chromatography binds to Millipore filters under conditions where other poly(A)-containing mRNAs have been shown to bind, and the subsequent finding that the bound and non-bound fractions contain different size classes of poly(A). The poly(A) size was determined by polyacrylamide gel electrophoresis of the T₁ and pancreatic RNAase-resistant fragments. The unbound mRNA fraction gives a fragment 35 to 45 adenine nucleotides long, while the bound mRNA contains two fragments with average lengths of 55 to 65 and 75 to 120 nucleotides.The heterogeneity of the poly(A) region is present in both α and β-globin mRNAs as both Millipore-bound and unbound RNA fractions directed the synthesis of comparable amounts of mouse α and β-globin chains.Change in the distribution of the various size classes of poly(A) was analyzed by Millipore binding assays after various times of labeling in vivo. The percentage of labeled mRNA bound to Millipore filters decreased with time, suggesting either a shortening of the poly(A) region or differential synthesis of mRNAs containing shorter poly(A) at earlier stages in erythropoeisis.     

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.     

The number of globin gene sequences in "cytoplasmic" DNA fragments.

DNA was isolated from the cytoplasm of primary cultures of mouse foetal liver cells. The proportion of globin genes was determined by two methods of cDNA-DNA hybridisation in globin complementary DNA excess. The proportion was similar in ‘cytoplasmic’ DNA and nuclear DNA. This argues against an informational role for this class of ‘cytoplasmic’ DNA, which has all of the characteristics of nuclear DNA arising from nucleosomes derived from chromatin.     

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences

Summary In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid or substitution loops due to insertion of globin DNA sequences combined with deletions of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.     

Absence from the mouse genome of DNA sequences related to the globin gene family.

Radioactively labelled DNA, complementary to mouse α and β globin messenger RNA, was annealed with unlabelled mouse embryo DNA under conditions of both optimum and lowered stringency. Although an increase in saturation of unlabelled DNA fragments with complementary DNA molecules is produced by lowered stringency, the values obtained are within the range expected for the known globin chains.It is concluded that within the limits of our experimental conditions, there does not exist a large family of DNA sequences related to the globin genes.     

The expression of viral and globin genes during differentiation of the friend cell

A complementary DNA probe has been prepared from the Friend murine erythroleukaemia virus complex (FV) released from Friend cells treated with dimethylsulphoxide (DMSO). The complementary DNA (cDNA) forms a hybrid specifically with the viral RNA genome. The availability of this viral probe together with mouse globin cDNA has made it possible to study the expression of both viral and globin genes in the Friend cell during differentiation using molecular hybridisation techniques. These specific probes have been used in an attempt to determine whether any connection exists between expression of Friend virus sequences and erythroid differentiation as measured by globin gene expression. A titration technique has been used to quantitate the levels of Friend viral- and globin-specific sequences in various Friend cell lines which differ in their ability to release Friend virus in response to DMSO although all produce haemoglobin under the same conditions. The results show: (a) that Friend cell lines unable to release virus nevertheless have a large pool of entire virus specific sequences in the polysomes; (b) an increase in virus release induced with DMSO is normally associated with a modest increase in viral sequence in the polysomes; (c) most cell lines show an early accumulation of viral and a later increase in globin mRNA sequences; (d) in an exceptional virus-negative, BUdR-resistant cell clone (B8/3), the accumulation of globin mRNA takes place very rapidly but there is no concomitant increase in viral RNA during differentiation.     

Host restriction of friend leukemia virus; fate of input virion RNA

Host restriction of oncogenesis by RNA tumor viruses may be studied in vitro by measuring the replication of the lymphatic leukemia component of the Friend Virus Complex (LLV-F) in either NIH-Swiss or Balb/C mouse embryo cells. These cells derive from mice differing at the Fv-1 locus, which controls the replication of all murine RNA leukemia viruses. Studies of early events in the replication of LLV-F were carried out by following the infection of permissive and restrictive mouse embryo cells by ³²P labeled LLV-F. ³²P labeled viral genome RNA rapidly becomes associated with cell nuclei and may be found integrated to the same extent with high molecular weight host DNA of either permissive or restrictive cells. These results suggest that Fv-1 mediated host restriction of LLV-F occurs at a step following integration of viral genome RNA into host DNA.Two other conclusions are suggested by these data. The nucleus appears to be the site of activation and synthesis of DNA of the infecting virus; and the “provirus”, at least transiently, is represented as an RNA-DNA hybrid molecule covalently integrated with host cell DNA.     

Evolutionary analysis of hepatitis C virus gene sequences from 1953

Reconstructing the transmission history of infectious diseases in the absence of medical or epidemiological records often relies on the evolutionary analysis of pathogen genetic sequences. The precision of evolutionary estimates of epidemic history can be increased by the inclusion of sequences derived from ‘archived’ samples that are genetically distinct from contemporary strains. Historical sequences are especially valuable for viral pathogens that circulated for many years before being formally identified, including HIV and the hepatitis C virus (HCV). However, surprisingly few HCV isolates sampled before discovery of the virus in 1989 are currently available. Here, we report and analyse two HCV subgenomic sequences obtained from infected individuals in 1953, which represent the oldest genetic evidence of HCV infection. The pairwise genetic diversity between the two sequences indicates a substantial period of HCV transmission prior to the 1950s, and their inclusion in evolutionary analyses provides new estimates of the common ancestor of HCV in the USA. To explore and validate the evolutionary information provided by these sequences, we used a new phylogenetic molecular clock method to estimate the date of sampling of the archived strains, plus the dates of four more contemporary reference genomes. Despite the short fragments available, we conclude that the archived sequences are consistent with a proposed sampling date of 1953, although statistical uncertainty is large. Our cross-validation analyses suggest that the bias and low statistical power observed here likely arise from a combination of high evolutionary rate heterogeneity and an unstructured, star-like phylogeny. We expect that attempts to date other historical viruses under similar circumstances will meet similar problems.     

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted β rabbit and α and β mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted α rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which a 3 kb deletion adjacent to the insertion site was observed.     

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.     

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.