Statistical analyses of developmental sequences: the craniofacial region in marsupial and placental mammals

Heterochrony is most often thought to involve changes in the rate of development or maturation (rate changes). However, heterochrony can also involve changes in the timing of specific developmental events relative to other events (sequence changes). Sequence changes have received much less attention than have changes in developmental rates, in part because few methods exist for comparing developmental sequences. Here, we present two methods to statistically evaluate developmental sequence changes. First, Kendall's coefficient of concordance (W) is used to quantify overall similarity of developmental sequences in two or more groups of organisms, and second, ANOVA is used to identify the individual events that differ most in their relative developmental timing. Computer simulation is used to control for the nonindependence of species. We examine the sequence of developmental events in the craniofacial region of marsupial and placental mammals. We conclude that the most important differences in development in the two clades relate to the relative sequence of development of the central nervous system and somatic elements of the craniofacial region. The rationale behind the methods and their limitations are discussed, and the results from this study are compared with a previous analysis.     

Comparative locomotor performance of marsupial and placental mammals

Marsupials are often considered inferior to placental mammals in a number of physiological characters. Because locomotor performance is presumed to be an important component of fitness, we compared marsupials and placentals with regard to both maximal running speeds and maximal aerobic speeds (=speed at which the maximal rate of oxygen consumption, VOlmax, is attained). Maximal aerobic speed is related to an animal's maximal sustainable speed, and hence is a useful comparative index of stamina.
Maximal running speeds of 11 species of Australian marsupials, eight species of Australian murid rodents, two species of American didelphid marsupials, and two species of American rodents were measured in the laboratory and compared with data compiled from the literature. Our values are greater than, or equivalent to, those reported previously. Marsupials and placentals do not differ in maximal running speeds (nor do Australian rodents differ from non-Australian rodents). Within these groups, however, species and families may differ considerably. Some of the interspecific variation in maximal running speeds is related to differences in habitat: species inhabiting open habitats (e.g. deserts) tend to be faster than are species from habitats with more cover, or arboreal species.
Maximal aerobic speeds (compiled from the literature) were higher in large species than in small species. However, marsupials and placentals show no general difference with regard to maximal aerobic speeds.
Maximal running speeds and maximal aerobic speeds for 18 species of mammals were not correlated, after correcting for correlations with body size. Thus, the fastest sprinters do not necessarily have high maximal aerobic speeds.     

Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans.

A 2 kb fragment was isolated from an Anacystis nidulans genomic DNA library by hybridization with synthetic oligonucleotide probes derived from the N-terminal amino acid sequence of Anacystis photolyase. This fragment contains a 1452 bp-long open reading frame encoding a polypeptide of 484 amino acids (Mr 54475). Antibodies raised against purified Anacystis photolyase reacted with extracts of cells harboring fused genes between lacZ of Escherichia coli and this gene. A 40.7% similarity was found between the deduced amino acid sequences of Anacystis and E. coli photolyases, notwithstanding the difference in chromophore structure.     

Differential distribution of the P1 and P2 protamine gene sequences in eutherian and marsupial mammals and a monotreme

At the protein level, the P1 protamine is the predominant form of mammalian protamine, present in all mammalian spermatozoa analyzed to date. An additional variant, the P2 protamine, has been detected only in spermatozoa of the mouse, hamster and human. Southern blot analysis of a group of restriction enzyme-digested mammalian DNAs has revealed the presence of sequences homologous to the P1 and the P2 mouse protamine genes in diverse species. In agreement with protein studies, nucleotide sequences homologous to the mouse P1 protamine cDNA are widespread, being present in the genomic DNAs of human, rat, dog, ram, horse, bull, hamster, baboon, flying fox (megabat), microbat, boar, North American opossum, and wallaby. Although we detect genomic sequences with strong homology to the mouse protamine 2 cDNA in rat and hamster, we also find weaker but reproducible hybridization to the genomic DNA of human, boar, dog, bull, microbat, wallaby, and platypus. With the exception of the human, the P2 protamine has not been detected in the spermatozoa of these latter species.     

Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.     

Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase.

The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.     

Cloning of the phr gene and amplification of photolyase in Escherichia coli.

A new technique is developed for physically enriching recombinant DNA molecules in an in vitro recombination mixture. UV-irradiation of the donor DNA before recombination enables photoreactivating enzyme (PRE) (deoxyribodipyrimidine photolyase, EC 4.1.99.3) to attach to the donor segments in recombinant molecules. This attached protein causes retention of the recombinant molecules on a nitrocellulose filter, while molecules not containing donor DNA pass through. The bound DNA is repaired of its UV damage and released for insertion into cells by exposure to photoreactivating light in situ, yielding approx. 350-fold enrichment. Although applicable to any gene, this procedure has been used in cloning the Escherichia coli phr gene itself, permitting 100-fold amplification of the gene product in vivo.     

Cloning and characterization of a class II DNA photolyase from Chlamydomonas

Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases. Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast. Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase. The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency. The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene. Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence. This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase.     

Assay of DNA photolyase activity in spinach leaves in relation to cell compartmentation-evidence for lack of DNA photolyase in chloroplasts

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

Mandible shape in marsupial and placental carnivorous mammals: a morphological comparative study using geometric morphometrics

We analysed mandible shape of the orders Dasyuromorpha, Didelphimorphia, and Carnivora using two‐dimensional geometric morphometrics, in order to explore the relationship between shape, size, and phylogeny. We studied 541 specimens, covering most of the genera of the terrestrial Carnivora (115 species) and a wide sample of marsupials (36 species). The observed shape variation had an ecological component. As an example, omnivorous carnivores have thick mandibles and large talonids in the carnassials, while hypercarnivores possess short mandibles and reduced talonids. There is also a discrimination between different taxonomic groups (i.e. marsupials and Carnivora), indicating some kind of constraint. Size explains a large percentage of total variance (large species had shorter and stronger mandibles, with anteriorly displaced carnassials), was significant when phylogeny was taken into account with a comparative method, but not when size and shape were optimized on the phylogeny. Carnivora presents a larger disparity and variation in body size, which could be related to the difference in teeth replacement. The optimization of mandible shape on the phylogenetic tree indicates that functional aspects, such as diet, are a key factor in the evolution of the carnivore mandible, but also that there is a phylogenetic pattern that cannot be explained by differences in diet alone. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 836–855.     

Cloning and nucleotide sequence of placental hGH-V cDNA

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.