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We are studying novel tocotrienols, which have a number of activities that might interfere with the formation of atherosclerotic plaques, including hypocholesterolemic, antioxidant, anti-inflammatory and antiproliferation effects. This study compared the effects of alpha-tocopherol, the tocotrienol-rich fraction (TRF(25)) and didesmethyl tocotrienol (d-P(25)-T3) of rice bran on the pathogenesis of atherosclerotic lesions in C57BL/6 apolipoprotein (apo)E-deficient (-/-) mice. These mice are an excellent model because they become hyperlipidemic even when they consume a low fat diet and they develop complex atherosclerotic lesions similar to those of humans. These compounds were also tested in wild-type C57BL/6 apoE (+/+) and (+/-) mice fed low or high fat diets. When a high fat diet was supplemented with alpha-tocopherol, TRF(25) or d-P(25)-T3 and fed to mice (+/+) for 24 wk, atherosclerotic lesion size was reduced 23% (P = 0.33), 36% (P = 0.14) and 57% (P < 0.02), respectively, and in mice (+/-) fed for 18 wk, lesions were reduced by 19% (P = 0.15), 28% (P < 0.01) and 33% (P < 0.005), respectively, compared with mice fed a control diet. A low fat diet did not cause atherosclerotic lesions in these mice. The low fat diet supplemented with TRF(25) or d-P(25)-T3 fed to apoE-deficient (-/-) mice for 14 wk decreased atherosclerotic lesion size by 42% (P < 0.04) and 47% (P < 0.01), respectively, whereas alpha-tocopherol supplementation resulted in only an 11% (P = 0.62) reduction. These results demonstrate the superior efficacy of tocotrienols compared with alpha-tocopherol. Although tocotrienols decreased serum triglycerides, total and LDL cholesterol levels, the decreases in atherosclerotic lesions seem to be due to the other activities. Serum tocol concentrations in various groups are also described. This is the first report of a significant reduction in the atherosclerotic lesion size in all three genotypes of apoE mice fed a novel tocotrienol (d-P(25)-T3) of rice bran. Dietary tocotrienol supplements may provide a unique approach to promoting cardiovascular health.

Rabbit alpha globin gene copies have been made, using reverse transcriptase and DNA polymerase I, and cloned in bacterial plasmids. Plasmid pHb72 has been shown to contain the alpha gene sequence by restriction enzyme analysis and nucleotide sequencing studies, and therefore has been approved for propagation under P2 plus EK1 conditions by the National REcombinant DNA Committee.

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolates varied by no more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

beta-Actin mutations in chemically transformed human cell lines have been associated with tumorigenicity, an association consistent with other evidence suggesting that altered cytoskeletal proteins may have an important role in cancer initiation or progression. From a human promyelocytic leukemia cell line, we have isolated a gamma-actin cDNA clone with amino acid substitutions in a region highly conserved in the many actins analyzed. To our knowledge, this is the first example of a variant gamma-actin in a human neoplasm. A separate finding from the analysis of this clone is that the gamma-actin 3'-untranslated region is among the most highly conserved of all 3'-untranslated sequences so far reported, but is entirely different from the beta-actin 3'-untranslated region. The high degree of evolutionary conservation suggests that the 3'-untranslated regions of these two mRNAs have important and distinct functional roles that were already fully differentiated more than 100 million years ago. Mutations affecting four major cytoskeletal components have now been identified in human neoplastic cells. These findings suggest that mutated cytoskeletal genes may be members of a class of oncogenes, fundamentally different from both the nuclear-acting (e.g., myc and simian virus 40 large tumor antigen) and growth factor/receptor/protein kinase-related (e.g., sis, erbB, and ras) types of oncogenes.

Recombinant DNA clones have been generated from mouse myeloma MOPC 21 immunoglobulin kappa light chain mRNA. Complementary DNA (cDNA) synthesized on kappa light chain mRNA by reverse transcriptase was made double stranded and inserted into the bacterial plasmid vector, pMB9. Approximately 70 tetracycline-resistant transformed colonies containing kappa light chain mRNA sequences were identified by colony hybridization. Five of these recombinant clones were selected and characterized. Three clones contain both kappa light chain constant and variable region sequences. Two of these three recombinant clones have been shown to include all of the kappa light chain constant and variable region coding sequences. Another of the five selected recombinant clones contain kappa light chain constant region sequences. The remaining characterized clone appears to be derived from sequences at the 5'-end of kappa light chain mRNA, possibly extending to the terminal cap structure.

The entire sequence of a 541 bp insert in recombinant plasmid pHb1003 has been determined. This plasmid, which was shown to carry a cloned cDNA copy of the chicken alpha-globin mRNA, contains the complete structural gene as well as 19 bp of the 5'-untranslated region and 99 bp of the 3'-untranslated region. This sequence may encode a non-adult alpha-globin gene, especially since the cDNA clones were generated from phenylhydrazine-induced, globin-specific mRNA extracted from anemic white leghorns. The possibility that this alpha-globin might represent a stress globin is considered.

The recombinant plasmid pH21-1 consists of mouse-derived complementary DNA (cDNA) in the E. coli plasmid pMB9. The mouse insertion has been completely sequenced, and encodes the CH3 domain and half the CH2 domain of the immunoglobulin gamma1 heavy chain. The predicted amino acid sequence differs at several positions from that previously published for this protein. The pattern of codon usage resembles that in some other eukaryotic messenger RNAs. A computer program has been used to predict the optimum secondary structure for the mRNA encoding the CH3 domain and the inter-domain junction.

DNA synthesized in vitro from a phage M13 template has been cleaved at either guanine, adenine, or cytosine residues by ribosubstitution techniques. Fingerprints of the fragments obtained suggest that DNA sequencing will be possible with this technique.

The complete nucleotide sequence of the N1 neuraminidase gene of influenza virus A/USSR/90/77 was determined. Comparison of its predicted amino acid sequence with other N1 and N2 neuraminidases indicates that the N1 neuraminidases share most of the antigenic determinants mapped on the N2 neuraminidase but display at least one additional potentially antigenic region probably as a result of intersubtypic differences in glycosylation.

The complete nucleotide sequence of the hemagglutinin gene of influenza virus A/USSR/90/77 was determined. Comparison of hemagglutinin amino acid sequences from H1 field strains revealed five potential antigenic sites. Four of these sites correspond to those observed for H3 hemagglutinins, whereas the fifth apparently derives from differences in the glycosylation patterns between subtypes.

A 169-nucleotide region from the rabbit beta globin gene has been sequenced by analysis of complementary DNA's cloned in bacterial plasmids. Comparison of these sequences with those established for this gene by other techniques provides evidence of a high degree of fidelity and allows the unambiguous establishment that these plasmids do not contain harmful sequences.

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32 P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T 1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.

We present a computer method utilizing published values for base pairing energies to compute the most energetically favorable secondary structure of an RNA from its primary nucleotide sequence. After listing all possible double-helical regions, every pair of mutally incompatible regions (whose nucleotides overlap) is examined to determine whether parts of those two regions can be combined by branch migration to form a pair of compatible new subregions which together are more stable than either of the original regions separately. These subregions are added to the list of base pairing regions which will compete to form the best overall structure. Then, a 'hyperstructure matrix' is generated, containing the unique topological relationship between every pair of regions. We have shown that the best structure can be chosen directly from this matrix, without the necessity of creating and examing every possible secondary structure. We have included the results from our solution of the 5S rRNA of the cyanobacterium Anacystis nidulans as an example of our program's capabilities.

The existence of non-random base pairing in T4-specific mRNA is supported by the nuclease digestion patterns of the messenger for phage T4 lysozyme. Under conditions which denature RNA, the nuclease nicked lysozyme mRNA recovers its activity. This behaviour can be explained by a model in which secondary structure controls initiation at existing ribosomal attachment sites.

Chicken globin cDNA was prepared from mRNA isolated from phenylhydrazine-treated chicken reticulocytes, and cloned into the tetracycline-resistance gene of plasmid pMB9. Restriction enzyme analysis indicates that there are at least two classes of clones isolated. Nucleotide sequence data have shown that these classes corresponded to the chicken α- and β-globin genes.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Deoxynucleotides have been incorporated into RNA synthesized in vitro by RNA polymerase with either double-stranded or single-stranded DNA as a template. By use of this technique to block or promote cleavage at a particular phosphodiester bond, a variety of specific cleavages may be obtained with the available ribonucleases and deoxyribonuclease I. These methods should greatly increase the ease and rapidity of nucleotide sequence determinations.

THE identification of a new chicken α-globin gene is reported here. Nucleotide sequence analysis of clone pHb1003 shows it to code for an α-subunit with an amino acid sequence different from those normally observed in the adult (the αA and αD). Characterisation of a second clone, pHb1010, carrying the same novel sequence, suggests that this mRNA sequence was present in substantial quantity after very young (2–3-week-old) chickens were treated with phenylhydrazine. The results are therefore similar to those reported previously for sheep and goats, where treatment with phenylhydrazine results in the turn-on of a new stress globin gene. As the ease of obtaining nucleated erythrocytes from avian systems facilitates many types of biochemical studies, we believe that this finding may provide an especially useful system in which to study the molecular events involved in globin gene switching.

DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene sequences.

Nucleotide sequence analysis and restriction endonuclease mapping have been used to characterize a cDNA copy of immunoglobulin MOPC 21 Kappa mRNA clones in the bacterial plasmid pMB9. Three regions of the inserted cDNA of plasmid pL21-1 have been sequenced and match the known protein sequence at amino acid residues 1-24, 128-138 and 171-179. With these sequences to provide absolute correlations between the restriction map and the structural gene sequence it has been possible to exactly deduce the positions of all 11 of the insert restriction sites mapped within the structural gene. The pL21-1 insert contains the complete variable and constant regions as well as parts of the 3' untranslated and polypeptide leader coding sequences.

The human hepatitis B virus (HBV) and the woodchuck hepatitis virus (WHV) are closely related by several criteria and belong to the same class of DNA viruses. The DNA genomes from these viruses are difficult to obtain in quantities required for biochemical analysis. We have, therefore, cloned these two DNAs in the vector lambda gtWES and subcloned into the kanamycin resistance plasmid pA01. Comparison of the recombinant DNAs with authentic viral DNAs by specific hybridization, size, and restriction enzyme analysis suggests that the recombinants contain the complete genome of each virus. The nominal size of the cloned HBV genome was 3150 base pairs, compared to 3200 base pairs for the cloned WHV genome. The small amount of nucleic acid homology previously reported between the HBV and WHV DNAs could be demonstrated between the cloned viral DNAs.

Ataxia-telangiectasia (AT) is a human autosomal recessive disorder of childhood1,2 characterized by: (1) progressive cerebellar ataxia with degeneration of Purkinje cells; (2) hypersensitivity of fibroblasts and lymphocytes to ionizing radiation3; (3) a 61-fold and 184-fold increased cancer incidence in white and black patients, respectively4; (4) non-random chromosomal rearrangements in lymphocytes; (5) thymic hypoplasia with cellular and humoral (IgA and IgG2) immunodeficiencies; (6) elevated serum level of alphafetoprotein; (7) premature ageing; and (8) endocrine disorders, such as insulin-resistant diabetes mellitus. A DNA processing or repair protein is the suspected common denominator in this pathology5. Heterozygotes are generally healthy; however, the sensitivity of their cultured cells to ionizing radiation is intermediate between normal individuals and that of affected homozygotes6. Furthermore, heterozygous females are at an increased risk of breast cancer7,8. These findings, when coupled with an estimated carrier frequency of 0.5–5.0%, suggest that (1) as many as one in five women with breast cancer may carry the AT gene7 and that (2) the increased radiation sensitivity of AT heterozygotes may be causing radiation therapists to reduce the doses of radiation used for treating cancer in all patients10. To identify the genetic defect responsible for this multifaceted disorder, and to provide effective carrier detection, we performed a genetic linkage analysis of 31 families with AT-affected members. This has allowed us to localize a gene for AT to chromosomal region 11q22-23.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.

The sequence of the highly repetitive satellite HS-β DNA fraction from kangaroo rat Dipodomys ordii was determined independently by RNA and DNA sequencing techniques. A basic iterated sequence of 10 nucleotides with several mutational variations was found. Base-composition data are consistent with the proposed sequence and revealed a high content of 5-methylcytosine. DNA and RNA sequencing techniques used gave identical results, showing that the fidelity of synthesis of riboguanidine-substituted DNA under our conditions is adequate for nucleotide sequence studies.

PROTEIN synthesis requires initiation of translation at the correct site on mRNA. Two initiation factors, A and C (ref. 1), or F1 and F2 (ref. 2), promote the ApUpG-dependent attachment of formylmethionyl-tRNA (fmet-tRNAf) to Escherichia coli ribosomes2–4, and account for initiation AUG triplets5,6. A mechanism must exist, however, for the discrimination between internal and initial methionine codons. In order to study the possible role of the third initiation factor, B, in this selection mechanism, we have examined the role of the three factors in the synthesis of lysozyme promoted by T4 mRNA7. We assumed that polypeptide synthesis as measured by amino-acid incorporation should not necessarily require correct initiation, while active enzyme formation should be highly dependent on this process. We have used the ratio of enzyme formed to valine incorporated as a measure of the proportion of polypeptide molecules correctly initiated.

HL-60 is a human promyelocytic cell line with the capability of differentiating in vitro to give neutrophils, macrophages, or eosinophils. We screened libraries of HL-60 cDNA clones representing different time points during these differentiation processes to isolate clones corresponding to mRNAs whose expression is regulated during terminal differentiation. Upon sequencing this group of regulated clones, one clone encoding the heavy subunit and two clones encoding the light subunit of human ferritin were identified by reference to published amino acid sequences. Southern blot analyses showed that these clones are encoded by distinct multigene families. These clones identify two mRNAs whose ratios vary in a complex manner during both neutrophil and macrophage differentiation.