A Phylogroup of the Siberian Chipmunk from Korea (Tamias sibiricus barberi) Revealed from the Mitochondrial DNA Cytochrome b Gene

In the full sequences of the mtDNA cytochrome b gene, 26 haplotypes (Tamias sibiricus barberi) from six localities of central and southern Korea were distinct from 21 haplotypes (Tamias sibiricus orientalis) from five localities of northeast China and Vladivostok, Russia. The average Tamura–Nei nucleotide distance between the subspecies (11.40%) and maximum infrasubspecific distances (3.74% and 4.72%) support the subspecies classification of T. s. barberi based on morphometric comparison. The 26 haplotypes of T. s. barberi were also distinct from 2 haplotypes of T. s. orientalis and Tamias sibiricus jacutensis from far-east Russia (average distance, 11.86%). Thus T. s. barberi constitutes a “phylogroup” (average nucleotide distance > 10%); analyses with nuclear genes of northeast Asian specimens, including North Korean ones, are necessary to clarify its taxonomic status. Furthermore, 49 haplotypes of T. sibiricus from eastern Asia differed from 19 haplotypes of another 18 Tamias spp. from America (weighted-average distance, 18.58%). T. sibiricus is, therefore, distinct enough to be recognized as a subgenus, Eutamias.     

Differential Selection of Cells with Proviral c-myc and c-erbB Integrations after Avian Leukosis Virus Infection

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3′ half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis.     

Identification of c-myc coding region determinant RNA sequences and structures cleaved by an RNase1-like endoribonuclease

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.     

Expression of c-myc is not critical for cell proliferation in established human leukemia lines

Background

A study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides.

Results

RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147–1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest.

Conclusions

We conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.     

Two Sympatric Phylogroups of the Chinese Water Deer (Hydropotes inermis) Identified by Mitochondrial DNA Control Region and Cytochrome b Gene Analyses

Mitochondrial DNA (mtDNA) control region (927 bp) and cytochrome b gene (1,140 bp) sequences of the Chinese water deer (Hydropotes inermis) from China and Korea were obtained to examine the taxonomic status of two subspecies, H. i. inermis from China and H. i. argyropus from Korea. Two sympatric mtDNA clades (a major clade from China and Korea and a minor clade from Korea) with an average genetic distance of 2.1% in the control region and 1.3% in the cytochrome b gene were detected. These findings are not consistent with the current classification by pelage color. We propose a reconsideration of the validity of the subspecies designation by the statistical comparison of morphological characters including body color. The major common mtDNA phylogroup in the two allopatric subspecies could be explained by the contiguous distribution of the Chinese water deer from east China to Korea until recent years. The restriction in the range and number of the Chinese subspecies after the last glacier might have caused the disappearance of the minor phylogroup in China. The taxonomic status of the two groups in Korea should be clarified using nuclear DNA marker analyses as well as morphological characters including pelage color.     

Narrow phylogeographic origin of five introduced populations of the Siberian chipmunk Tamias (Eutamias) sibiricus (Laxmann, 1769) (Rodentia: Sciuridae) established in France

Large native geographical range and number of introduction events are often invoked to explain the successful establishment of alien mammal species. To infer the native geographic range of the Siberian chipmunks Tamias (Eutamias) sibiricus (Lax.) invasion in France, we identified the subspecies that individuals sampled in 5 out of the 11 established populations belonged. Based on craniometrical measures, variation in the mtDNA sequence of the cytochrome b, and alarm call sonogram shape, all French specimens were members of T. (E.) s. barberi which has the smallest geographical range (Korean peninsula) of the three recognized subspecies. An intense pet trade between European countries and Korea until the 1980’s best explains the success in establishment of this small ground-squirrel in France, where it was released mainly by private owners. Size of the native geographical range should be interpreted with caution in explaining the establishment of an invasive species, at least without a precise knowledge of its introduction pathways and taxonomic status.     

Activation of c-myc Gene Expression by Tumor-Derived p53 Mutants Requires a Discrete C-Terminal Domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3′ end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Identification,genetic variation,and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

The amplification of oligonucleotide themes in the evolution of themyc protooncogene family

The evolutionary past of intragenic repeats in protein-coding exons of c-, N-, L-, and s-myc-protooncogene subfamilies was elucidated. Apparently these genes evolved by succession of distinct unit events rather than by a steady flow of random point mutations. An evolutionary event probably involved a duplication of the whole gene, which was followed by amplification of progressively shorter oligonucleotide themes and motifs. The repeats were either joined in tandem or one of the copies was transposed and integrated elsewhere within the same exon. In some instances multiple fragments of an amplified theme were integrated at several sites. Direct repeats were found to prevail over inverted ones. By reconstructing the fate of repeats in the course of evolution of vertebrates, the origins of some functional domains could be traced to the initial amplification event. For example, an N-myc-specific domain was created by tandem duplication of a single-copy theme of L-myc exon; at the time of divergence of the c-myc and N-myc, the tandem duplex underwent a new round of duplication followed by transposition of the new copy, thus accounting for the formation of a new domain specific for c-myc. The model proposed here may be regarded as a molecular-level equivalent of the theory of punctuated equilibria. This is the author's last paper; it was submitted shortly before his death. The proofs were corrected by Michal Dvorak Correspondence to: M. Dvorak     

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.     

A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells

We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10⁵) for any target sequence for use in human cells. In this system, target sequences are fused to the 5′ end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc–lacZ fusion gene and the endogenous c-myc gene in human cells.