High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.     

Episodic evolution mediates interspecies transfer of a murine coronavirus.

Molecular mechanisms permitting the establishment and dissemination of a virus within a newly adopted host species are poorly understood. Mouse hepatitis virus (MHV) strains (MHV-A59, MHV-JHM, and MHV-A59/MHV-JHM) were passaged in mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine DBT cells. From MHV-A59/MHV-JHM mixed infection, variant viruses (MHV-H1 and MHV-H2) which replicated efficiently in BHK cells were isolated. Under identical treatment conditions, the parental MHV-A59 or MHV-JHM strains failed to produce infectious virus or transcribe detectable levels of viral RNA or protein. The MHV-H isolates were polytrophic, replicating efficiently in normally nonpermissive Syrian hamster smooth muscle (DDT-1), Chinese hamster ovary (CHO), human adenocarcinoma (HRT), primate kidney (Vero), and murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK) and porcine testicular (ST) cell lines. The variant virus, MHV-H2, transcribed seven mRNAs equivalent in relative abundance and size to those synthesized by the parental virus strains. MHV-H2 was an RNA recombinant virus containing a crossover site in the S glycoprotein gene. At the molecular level, episodic evolution and positive Darwinian natural selection were apparent within the MHV-H2 S and HE glycoprotein genes. These findings differ from the hypothesis that neutral changes are the predominant feature of molecular evolution and argue that changing ecologies actuate episodic evolution in the MHV spike glycoprotein genes that govern interspecies transfer and spread into alternative hosts.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.     

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Lake level fluctuations synchronize genetic divergences of cichlid fishes in African lakes

Water level fluctuations are important modulators of speciation processes in tropical lakes, in that they temporarily form or break down barriers to gene flow among adjacent populations and/or incipient species. Time estimates of the most recent major lowstands of the three African Great Lakes are thus crucial to infer the relative timescales of explosive speciation events in cichlid species flocks. Our approach combines geological evidence with genetic divergence data of cichlid fishes from the three Great East African Lakes derived from the fastest-evolving mtDNA segment. Thereby, we show for each of the three lakes that individuals sampled from several populations which are currently isolated by long geographic distances and/or deep water form clusters of equally closely related haplotypes. The distribution of identical or equally closely related haplotypes in a lake basin allows delineation of the extent of lake level fluctuations. Our data suggest that the same climatic phenomenon synchronized the onset of genetic divergence of lineages in all three species flocks, such that their most recent evolutionary history seems to be linked to the same external modulators of adaptive radiation. A calibration of the molecular clock of the control region was elaborated by gauging the age of the Lake Malawi species flock through the divergence among the utaka-cichlid and the mbuna-cichlid lineages to minimally 570,000 years and maximally 1 Myr. This suggests that the low-lake-level period which established the observed patterns of genetic relatedness dates back less than 57,000 years, probably even to 17,000-12,400 years ago, when Lake Victoria dried up and Lakes Malawi and Tanganyika were also low. A rapid rise of all three lakes about 11,000 years ago established the large-scale population subdivisions observed today. Over that period of time, a multitude of species originated in Lakes Malawi and Victoria with an impressive degree of morphological and ecological differentiation, whereas the Tanganyikan taxa that were exposed to the same habitat changes hardly diverged ecologically and morphologically. Our findings also show that patterns of genetic divergences of stenotopic organisms provide valuable feedback on geological and sedimentological time estimates for lake level changes.