Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

A genetic analysis of natural resistance to nonsyngeneic cells: The role of H-2

The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2 ^dtumor cells and tumor cell elimination was lowest in H-2 ^dhomozygotes. Natural killer cell activity was also reduced in mice with the H-2 ^dhaplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2 ^dtumors was restricted to H-2 ^bstrains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2 ^dand H-2 ^btumors in DBA/2, C57BL/6, B6D2F₁, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin.     

The formation of supernumerary structures after grafting anterior quail wing bud mesoderm of various ages into chick wing buds

Wedges of anterior quail mesoderm grafted into posterior slits in the wing buds of chick embryo hosts result in the formation of rods and nodules of supernumerary cartilage in a high percentage of cases. Identifiable digits do not form unless the ectoderm is allowed to remain on the grafts. Control experiments have shown that wedges of anterior or posterior wing mesoderm placed into homologous locations of host wing buds produce few or no supernumerary skeletal structures. Anterior-to-posterior grafts of stage 17 mesoderm evoke a 71.4% incidence of supernumerary cartilage. This percentage increases to 100% with stage 22 donor mesoderm. The percentage of supernumerary structures formed declines markedly with donor mesoderm of stages 24-30. By stages 35-36, only 10% of the grafts result in the formation of supernumerary structures. The period of decline coincides with the onset of overt cytodifferentiation within the donor mesoderm.     

The preservation of the ability of cultured quail wing bud mesoderm to elicit a position-related differentiative response

A previous study showed that grafting wedges of fresh anterior quail wing mesoderm into posterior slits of chick wing buds resulted in the formation of rods and nodules of cartilage in a high percentage of cases (B. Carlson, 1983, Dev. Biol. 101, 97-105). The purpose of the present study was to determine if a similar response could be elicited by grafting pieces of mesoderm that had been cultured in vitro. When pieces of 1-day cultured anterior mesoderm from stage 17-24 donors were grafted into standard posterior slits of chick wing buds, the percentages of supernumerary structures differed little from those which formed after the grafting of pieces of fresh mesoderm. In a time series, grafts of stage 22-23 anterior mesoderm which had been cultured for 1-4 days retained the ability to form cartilage after being grafted into posterior locations. A time series showed that the duration of this retention was longer in cultured mesoderm than it was in mesoderm that remains in the donor wing bud.     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus

We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported.     

Anaphase chromosome movement in the unequally dividing grasshopper neuroblast and its relation to anaphases of other cells

The positions of the two sets of chromosome kinetochores, the spindle poles, cell membrane adjacent to the poles, and cleavage furrow of grasshopper neuroblasts in culture at 38°C were determined at short-time intervals during anaphase. The percent of motion due to poleward movement and spindle elongation, which coincide in time, were calculated for each minute, the former falling from 61% in the first minute to 15% in the seventh minute, and increasing to 86% in the final minute, probably as a result of pressure and bending of the spindle. Of the total chromosome movement during anaphase 44.6% is due to poleward movement of the daughter kinetochores and 55.4% to spindle elongation. The maximum velocity of a set of kinetochores is 3.41 m/min and the mean velocity 1.86 m/min (one-half the rate of separation). Various studies of anaphase chromosome movement in different cells and different species suggest certain generalizations, some of which are based on very small samples and so must be considered quite tentative: (1) The combination of poleward movement and spindle elongation is much more frequent than either acting alone. (2) These components of movement may coincide in time, overlap, or spindle elongation may follow poleward movement, but spindle elongation never begins before poleward chromosome movement. (3) There is an optimum temperature for the rate of chromosome movement, above and below which the rate gradually decreases. (4) In homoiothermic animals this optimum occurs at normal body temperature. (5) In homoiothermic animals the velocity falls more rapidly with a decrease in temperature than in poikilothermic animals. (6) Animals with large chromosomes (amphibia, grasshoppers) have higher chromosome velocities than those with small chromosomes. (7) Non-meiotic cells and secondary spermatocytes have higher velocities than primary spermatocytes of the same species. (8) Chromosome velocity is lower in malignant than non-malignant cells. (9) Chromosome velocity tends to be positively correlated with the distance the chromosomes travel during anaphase.     

Prey capture in mantids: Prothoracic tibial flexion reflex

We describe a prothoracic leg tibial flexion reflex (PTFR) of the praying mantis Tenodera aridifolia sinensis which is initiated by tactile stimulation of the movable spines of the ventro-medial border of the femur. This flexion reflex may be responsible for the continuous grasping of a captured prey by the mantid.