Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Dynamic domains of gene expression in the early avian forebrain.

The expression domains of genes implicated in forebrain patterning often share borders at specific anteroposterior positions. This observation lies at the heart of the prosomeric model, which proposes that such shared borders coincide with proposed compartment boundaries and that specific combinations of genes expressed within each compartment are responsible for its patterning. Thus, genes such as Emx1, Emx2, Pax6, and qin (Bf1) are seen as being responsible for specifying different regions in the forebrain (diencephalon and telencephalon). However, the early expression of these genes, before the appearance of putative compartment boundaries, has not been characterized. In order to determine whether they have stable expression domains before this stage, we have compared mRNA expression of each of the above genes, relative both to one another and to morphological landmarks, in closely staged chick embryos. We find that, between HH stage 8 and HH stage 13, each of the genes has a dynamic spatial and temporal expression pattern. To test for autonomy of gene expression in the prosencephalon, we grafted tissue from this region to more caudal positions in the neural tube and analyzed for expression of Emx1, Emx2, qin, or Pax6. We find that gene expression is autonomous in prosencephalic tissue from as early as HH stage 8. In the case of Emx1, our data suggest that, from as early stage 8, presumptive telencephalic tissue also is committed to express this gene. We propose that early patterning along the anteroposterior axis of the presumptive telencephalon occurs across a field that is subdivided by different combinations of genes, with some overlapping areas, but without either sharp boundaries or stable interfaces between expression domains.     

Phaseshifting of the photoperiodic flowering response rhythm in Pharbitis nil by red-light pulses

The control by light of the flowering response rhythm in the short-day plant Pharbitis nil Choisy cv. Violet was examined by giving a single pulse of light at various times between 1 and 6 h after a 24-h light period. When the first circadian cycle of the rhythm was monitored, it was found that a pulse of red light given at 1, 2 or 3 h into a 72-dark period caused a 1-h delay of the phase of the response rhythm, while a pulse at 6 h caused a 2-h delay. These results support the hypothesis that, when red-light pulses are given at hourly intervals, they are as effective as continuous light in preventing the onset of dark timing because they repeatedly return the rhythm to the circadian time at which it is apparently suspended in continuous light. The perception of and response to continuous light and red-light pulses are also briefly discussed.     

The role of the notochord for epaxial myotome formation in the mouse.

The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals.     

Specification of distinct motor neuron identities by the singular activities of individual Hox genes.

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. We have employed a direct gain-of-function approach using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though it is normally devoid of this cell type. These ectopic neurons have the specific identity of branchiomotor neurons and, in the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. Next, we have attempted to identify the precise branchiomotor subtypes that are generated after misexpression and our results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. Our data demonstrate, therefore, that at least to a certain extent and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, as these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, our data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance.