Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified.

Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Induction of a G2-Phase Arrest in Xenopus Egg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.

Characterization of Saccharomyces cerevisiae dna2 Mutants Suggests a Role for the Helicase Late in S Phase

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

A novel host factor for integration of mycobacteriophage L5

Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase–mIHF–attP intasome complexes.

Biodiversity of Costa Rican salamanders: Implications of high levels of genetic differentiation and phylogeographic structure for species formation

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display “tropicality” in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.

Population genetics of Ice Age brown bears

The Pleistocene was a dynamic period for Holarctic mammal species, complicated by episodes of glaciation, local extinctions, and intercontinental migration. The genetic consequences of these events are difficult to resolve from the study of present-day populations. To provide a direct view of population genetics in the late Pleistocene, we measured mitochondrial DNA sequence variation in seven permafrost-preserved brown bear (Ursus arctos) specimens, dated from 14,000 to 42,000 years ago. Approximately 36,000 years ago, the Beringian brown bear population had a higher genetic diversity than any extant North American population, but by 15,000 years ago genetic diversity appears similar to the modern day. The older, genetically diverse, Beringian population contained sequences from three clades now restricted to local regions within North America, indicating that current phylogeographic patterns may provide misleading data for evolutionary studies and conservation management. The late Pleistocene phylogeographic data also indicate possible colonization routes to areas south of the Cordilleran ice sheet.

Formation of the preprimosome protects λ O from RNA transcription-dependent proteolysis by ClpP/ClpX

Using the bacteriophage λ DNA replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived λ O protein to the ClpP/ClpX protease during the various stages of λ DNA replication. We find that binding of λ O protein to its oriλ DNA sequence, leading to the so-called “O-some” formation, largely inhibits its degradation. On the contrary, under conditions permissive for transcription, the λ O protein bound to the oriλ sequence becomes largely accessible to ClpP/ClpX-mediated proteolysis. However, when the λ O protein is part of the larger oriλ:O⋅P⋅DnaB preprimosomal complex, transcription does not significantly increase ClpP/ClpX-dependent λ O degradation. These results show that transcription can stimulate proteolysis of a protein that is required for the initiation of DNA replication.

Fetal and maternal contributions to risk of pre-eclampsia: population based study

Objective: To use familial patterns of recurrence of pre-eclampsia to investigate whether paternal genes expressed in the fetus contribute to the mother’s risk of pre-eclampsia and whether mother’s susceptibility to pre-eclampsia is related to maternal inheritance by mitochondrial DNA.

Replicative helicases can translocate through abasic site-induced covalent topoisomerase IV–DNA complexes

Some topoisomerase inhibitors trap covalent topoisomerase–DNA complexes as topoisomerase–drug–DNA ternary complexes. Ternary complex formation results in inhibition of DNA replication and generation of permanent double-strand breaks. Recent demonstrations of the stimulation of covalent topoisomerase–DNA complex formation by DNA lesions suggest that DNA damage may act as an endogenous topoisomerase poison. We have investigated the effects of abasic (AP) sites on topoisomerase IV (Topo IV). AP sites can stimulate the formation of covalent Topo IV–DNA complexes when they are located either within the 4 base overhang generated by DNA scission or immediately 5′ to the point of scission (the –1 position). Thus, the AP site acts as a position-specific, endogenous topoisomerase poison. Both EDTA and salt can reverse covalent Topo IV–DNA complexes induced by AP sites located within the 4 base overhang. Interestingly, an AP site at the –1 position inhibits EDTA-mediated reversal of formation of the covalent Topo IV–DNA complex. Furthermore, we find that, unlike quinolone-induced covalent Topo IV–DNA complexes, AP site-induced covalent Topo IV–DNA complexes do not inhibit the helicase activities of the DnaB and T7 Gene 4 proteins. These results suggest that the AP site-induced poisoning of Topo IV does not arrest replication fork progression.

Positive Regulation of Wee1 by Chk1 and 14-3-3 Proteins

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.