Sequence homology between mitochondrial DNAs of different eukaryotes.

The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization. mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA. Single-stranded and duples DNA were separated by hydroxylapatite chromatography. The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns. Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken. Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess. Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex. There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to [3H]mtDNA was varied between 1250 and 7500. Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation. Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation. Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.     

The complete mitochondrial DNA sequence of the harbor seal,Phoca vitulina

Summary The nucleotide sequence of the mitochondrial DNA (mtDNA) of the harbor seal, Phoca vitulina, was determined. The total length of the molecule was 16,826 bp. The organization of the coding regions of the molecule conforms with that of other mammals, but the control region is unusually long. A considerable portion of the control region is made up of short repeats with the motif GTACAC particularly frequent. The two rRNA genes and the 13 peptide-coding genes of the harbor seal, fin whale, cow, human, mouse, and rat were compared and the relationships between the different species assessed. At ordinal level the 12S rRNA gene and 7 out of the 13 peptide-coding genes yielded a congruent topological tree of the mtDNA relationship between the seal, cow, whale, human, and the rodents. In this tree the whale and the cow join first, and this clade is most closely related to the seal.Offprint requests to: Ú. Árnason     

Regional localization of the fibronectin and gamma-crystallin genes to bovine chromosome 8

The genes for isocitrate dehydrogenase-1, fibronectin, and gamma-crystallin are syntenic in man, mouse, and cow. In an effort to assign this bovine syntenic group to a specific chromosome and to allow a cytological comparison of the conserved chromosomal region containing these genes in their respective species, we have localized the fibronectin and gamma-crystallin genes to bovine chromosome 8, region 1.1-1.4. This study incorporates the techniques of hybrid somatic cell analysis and in situ hybridization and the use of a Robertsonian-translocated marker chromosome from a related species for regional assignment of genes to a specific bovine chromosome. The regions on human chromosome 2q, mouse chromosome 1, and cow chromosome 8 that contain these genes are cytologically similar, perhaps representing evolutionary conservation at the cytogenetic level as well as at the gene level for this group of loci.     

Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10(-4)) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Peptide-containing nerve fibers in the circumvallate papillae.

The occurrence and distribution of an array of neuropeptides and dopamine-beta-hydroxylase in the circumvallate papillae of monkey, pig, cow, ferret, cat, rat and mouse was studied by immunocytochemistry. The animals were chosen to represent species with different diets. Substance P/neurokinin A- and calcitonin gene-related peptide-containing fibers were numerous in the circumvallate papillae of all animals examined, with the highest frequency in monkey, pig, cow, rat and mouse; in ferret and cat moderate numbers were detected. Vasoactive intestinal peptide/peptide histidine isoleucine amide-containing fibers were numerous in the circumvallate papillae of pig, while they were moderate in number in monkey, ferret and mouse. Neuropeptide Y-containing fibers were few to moderate in number in the circumvallate papillae of all species. Galanin-containing fibers were numerous in the pig circumvallate papillae, while only a few fibers could be detected in monkey, cow, cat, rat and mouse. Somatostatin-containing fibers were seen only in the cat circumvallate papillae, gastrin-releasing peptide-containing fibers in the cow and cat, cholecystokinin/gastrin-containing fibers in the pig and cow. Dopamine-beta-hydroxylase-containing fibers were detected in all animals studied. They were few to moderate in number in the circumvallate papillae. There was no obvious link between the peptidergic innervation pattern and the food habits.     

Co-linear organization of Xenopus laevis and mouse mitochondrial genomes

Cloned fragments of Xenopus laevis mitochondrial DNA and Pleurodeles waltlii mitochondrial cDNAs have been hybridized together and with mouse mtDNA. In the three cases cross-hybridization was observed. The overall organization of the X. laevis fragments appeared to be co-linear with the mouse mtDNA, most sequences being conserved except for the D-loop and the URF6 regions. The use of mouse mtDNA has enabled us to identified several mitochondrial genes in X. laevis and P. waltlii.     

One-Step Lipophilization of Proteins Using Fatty Acyl Anhydride

Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5′- and 3′-untranslated regions, respectively. Homology in the comparable coding region of the mature asl-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.     

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.     

Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.     

Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects

The production of in vitro and in vivo models of mitochondrial DNA (mtDNA) defects is currently limited by a lack of characterized mouse cell mtDNA mutants that may be expected to model human mitochondrial diseases. Here we describe the creation of transmitochondrial mouse (Mus musculus) cells repopulated with mtDNA from different murid species (xenomitochondrial cybrids). The closely related Mus spretus mtDNA is readily maintained when introduced into M. musculus mtDNA-less (rho(0)) cells, and the resulting cybrids have normal oxidative phosphorylation (OXPHOS). When the more distantly related Rattus norvegicus mtDNA is transferred to the mouse nuclear background the mtDNA is replicated, transcribed, and translated efficiently. However, function of several OXPHOS complexes that depend on the coordinated assembly of nuclear and mtDNA-encoded proteins is impaired. Complex I activity in the Rattus xenocybrid was 46% of the control mean; complex III was 37%, and complex IV was 78%. These defects combined to restrict maximal respiration to 12-31% of the control and M. spretus xenocybrids, as measured polarographically using isolated cybrid mitochondria. These defects are distinct to those previously reported for human/primate xenocybrids. It should be possible to produce other mouse xenocybrid constructs with less severe OXPHOS phenotypes, to model human mtDNA diseases.     

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.     

A comparison of the structure and properties of serum transferrin from 17 animal species

1. A comparison of the chemical and physical properties of the iron transport protein transferrin, purified from the following seventeen animal sera, is reported; human, rhesus monkey, dog, cat, rabbit, guinea pig, mouse, rat, cow, sheep, goat, horse, pig, turkey, duck, turtle and rattlesnake. 2. Similarities and differences in molecular weight, isoelectric point, antibody specificity, effect of pH on iron release, number of sialic acid residues, amino acid composition and the N-terminal amino acid residue, are discussed. 3. The results are compared with the commonly accepted evolutionary origins of the 17 species.     

A comparison of myosin light chain subunits in the atria and ventricles of mammals

An SDS-electrophoretic comparison of atrial and ventricular myosin light chain isotypes was performed in mouse, rat, rabbit, dog, pig, rhesus monkey, baboon, human and cow heart. Light chains 1 and 2 in atria and ventricles differed in all species with the possible exception of the rhesus monkey. Relative migration of atrial and ventricular LC-2 isotypes was similar in all species but LC-1 isotypes varied in relative migration rates suggesting increased primary sequence heterogeneity. Order of migration was VLC-1 less than ALC-1 less than ALC-2 less than VLC-2 in mouse, rat, rabbit, dog, baboon and cow and ALC-1 less than VLC-1 less than ALC-2 less than VLC-2 in pig and human heart. No obvious relationship existed between electrophoretic pattern and phylogenetic evolution.     

Extensive diversity among Drosophila species with respect to nucleotide sequences within the adenine + thymine-rich region of mitochondrial DNA molecules.

Mitochondrial DNA (mtDNA) molecules from species of the genus Drosophila contain a region exceptionally rich in adenine + thymine (A+T). Using agarose gel electrophoresis and electron microscopy, we determined that in the mtDNA molecules of D. melanogaster, D. simulans, D. mauritiana, D. yakuba, D. takahashii, and D. virilis, the A+T-rich regions, which are 5.1, 4.8, 4.6, 1.1, 2.2, and 1.0 kilobase pairs in size, respectively, are at homologous locations relative to various common EcoRI and HindIII cleavage sites. Under conditions highly permissive for base pairing (35% formamide), heteroduplexes were constructed between EcoRI fragments and whole circular molecules of mtDNAs of the above mentioned six species in a variety of combinations. Complete pairing of molecules outside the A+T-rich region was found in all heteroduplexes examined. However, in contrast, A+T-rich regions of the different species failed to pair in all but those combinations of mtDNAs involving the three most closely related species. In heteroduplexes between D. melanogaster and D. simulans, and between D. melanogaster and D. mauritiana mtDNAs, up to 35% of the A+T-rich regions appeared double-stranded. These data indicate that much more extensive divergence of sequences has occurred in A+T-rich regions than in other regions of Drosophila mtDNA molecules.     

An electron microscopic heteroduplex study of the sequence relations between the bacteriophages LP52 and theta

Summary The genomes of the phylogenetically related but morphologically distinct bacteriophages LP52 and theta () were compared by electron microscopic heteroduplex analysis. The heteroduplex maps were aligned with known restriction maps. In the heteroduplices of LP52 DNA (63.8 kb) with the DNA of the lytic phage _c (65.9 kb) the tracts of homologous DNA cover about 50% of the genome length and are interspaced by four large and ten smaller non-base-paired regions. The largest block of non-homologous DNA (18.9 kb), represents the right-hand end and there is an unmatched piece of DNA at the left-hand end as well. Most of the heterology is due to substitution resulting in the conservation of the total length of DNA; the three insertions/deletions amount to less than 3.2% of the genome length. Heteroduplices between the DNAs of phage LP52 and the temperate phage ₁ (65.0 kb) resembled those of LP52: _c except for the absence of minor loops. Heteroduplex _c:₁ displayed about 9% heterology in seven separate loops which coincided with sections of diversity on the restriction maps; 4.8% of ₁ DNA did not hybridize with either _c or LP52 DNA.