Brachyury (T) Expression in Embryos of a Larvacean Urochordate, Oikopleura dioica, and the Ancestral Role of T

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate–vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord.     

Origin of Chordata and the “Upside-Down Theory”

The hypothesis of dorso-ventral axis inversion in Chordata based on recent molecular biology data is discussed from the viewpoint of evolutionary morphology. It is supposed that, as a result of such an inversion, the nerve cord located ventrally in most Invertebrata found itself on the dorsal side of Chordata. However, an analysis of the contemporary hypotheses explaining chordate origin (dipleuruloid and hemichordate) shows that neither of them provides convincing evidence of dorso-ventral inversion. A new hypothesis agreeing with both the molecular and morphological data is suggested. According to this hypothesis, the divergence of Protostomia and Deuterostomia occurred early in the origin of Bilateria. The initial stage (for a common ancestor) is assumed to be some turbellarians, in which several nerve cords extend back from the apical organ in a radial-symmetrical pattern. In the process of subsequent oligomerization, the ventral nerve cords become more developed in Protostomia, while in Deuterostomia the dorsal nerve cords have priority. It is emphasized that reaching an understanding between molecular biologists and evolutionary morphologists is a necessity.     

Evolution of circular permutations in multidomain proteins

Modular rearrangements play an important role in protein evolution. Functional modules, often tantamount to structural domains or smaller fragments, are in many cases well conserved but reoccur in a different order and across many protein families. The underlying genetic mechanisms are gene duplication, fusion, and loss of sequence fragments. As a consequence, the sequential order of domains can be inverted, leading to what is known as circularly permutated proteins. Using a recently developed algorithm, we have identified a large number of such rearrangements and analyzed their evolutionary history. We searched for examples which have arisen by one of the three postulated mechanisms: independent fusion/fission, "duplication/deletion," and plasmid-mediated "cut and paste." We conclude that all three mechanisms can be observed, with the independent fusion/fission being the most frequent. This can be partly attributed to highly mobile domains. Duplication/deletion has been found in modular proteins such as peptide synthases.     

The urochordate larva and archichordate organization: chordate origin and anagenesis revisited†

A critical analysis of relevant characters in Epineuralia (Tentaculata + Deuterostomia) in order to trace the emergence of chordate organization demonstrates that a reorganization of already highly adapted planktotrophic larvae is inconceivable. In connection with the clearly regressive state of urochordate organization, an alternative concept is presented. It proposes the chordate origin via adaptation of simply ciliated, short-ranged lecithotrophic *hemichordate* larvae which retained a dorsoposterior primary anus (see Pterobranchia, Enteropneusta-Harrimaniidae, lower Chordata). The extension of larval life to exploit the food-rich layers of the sea enabled the posterior, pre-anal body (rather than a ‘tail’) to develop into a powerful propulsive complex. This was prepared by a mesoblastema heterochrony and was co-evolutively accompanied by (1) the precocious differentiation of the middorsal nerve cord of *Hemichordata* to become a neural plate extending onto the metasoma (meeting the dorsoposterior anus: neurenteric canal) (2) a cerebral sensory centre, and (3) the precociousness of the filter-feeding branchial gut. Such larval, primitive Archichordata were coupled in their life-cycle most probably with semi-vagile *hemichordate* adults (lophophore already reduced; with new endostyle). This biphasic organization subsequently radiated into the two clades of (1) Tunicata (elaboration of the branchial gut with sedentary life, peribranchial cavity, tunica/cuticle, and regressive larvae; secondarily neotene and or pelagic) and (2) Holochordata (prematurity of larvae with extension of the somatic chordate organization forwards over the branchial gut). The latter level gave rise to (a) the Acrania (Cephalochordata) turning to an epibenthic life (fairly stationary habit with asymmetry, regression of the cerebral sensory equipment) and finally becoming infaunal: (b) the Craniata (Vertebrata) elaborating the pelagic existence and changing to a macrophagous biology (selective particle-swallowing rather than mucociliary filter-feeding), correlated with a highly adapted sensory control system (cephalization) and neural crest organization.     

Evolutionary reorganizations of ontogenesis in ascidians of the genus Molgula

The data on comparative, experimental, and molecular embryology of ascidians (genus Molgula) published during the last 15 years have been reviewed. Some representatives of this genus evolved from development with a tailed larva (tadpole) to direct development associated with the loss of larval structures, such as tail, notochord, sensory organs, and differentiated muscles. The data on evolutionary reorganizations of ontogenesis in ascidians of the genus Molgula have been compared with those in sea urchins, anuran amphibians, and some other organisms.     

Strategies for maximizing metallothionein promoter regulated recombinant protein production in mammalian cell cultures

A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn⁺² or Cd⁺²) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd⁺² dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd⁺² although cell-associated Cd⁺² levels remain high. Further addition of Cd⁺² to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd⁺² is sequestered into a form which does not stimulate the MT promoter. Cd⁺² dosing regimes which maintained cell associated Cd⁺² concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd⁺² loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line.     

Amyloid cores in prion domains: Key regulators for prion conformational conversion

Despite the significant efforts devoted to decipher the particular protein features that encode for a prion or prion-like behavior, they are still poorly understood. The well-characterized yeast prions constitute an ideal model system to address this question, because, in these proteins, the prion activity can be univocally assigned to a specific region of their sequence, known as the prion forming domain (PFD). These PFDs are intrinsically disordered, relatively long and, in many cases, of low complexity, being enriched in glutamine/asparagine residues. Computational analyses have identified a significant number of proteins having similar domains in the human proteome. The compositional bias of these regions plays an important role in the transition of the prions to the amyloid state. However, it is difficult to explain how composition alone can account for the formation of specific contacts that position correctly PFDs and provide the enthalpic force to compensate for the large entropic cost of immobilizing these domains in the initial assemblies. We have hypothesized that short, sequence-specific, amyloid cores embedded in PFDs can perform these functions and, accordingly, act as preferential nucleation centers in both spontaneous and seeded aggregation. We have shown that the implementation of this concept in a prediction algorithm allows to score the prion propensities of putative PFDs with high accuracy. Recently, we have provided experimental evidence for the existence of such amyloid cores in the PFDs of Sup35, Ure2, Swi1, and Mot3 yeast prions. The fibrils formed by these short stretches may recognize and promote the aggregation of the complete proteins inside cells, being thus a promising tool for targeted protein inactivation.     

Prediction of the location of structural domains in globular proteins

The location of structural domains in proteins is predicted from the amino acid sequence, based on the analysis of a computed contact map for the protein, the average distance map (ADM). Interactions between residues i and j in a protein are subdivided into several ranges, according to the separation |i-j| in the amino acid sequence. Within each range, average spatial distances between every pair of amino acid residues are computed from a data base of known protein structures. Infrequently occurring pairs are omitted as being statistically insignificant. The average distances are used to construct a predicted ADM. The ADM is analyzed for the occurrence of regions with high densities of contacts (compact regions). Locations of rapid changes of density between various parts of the map are determined by the use of scanning plots of contact densities. These locations serve to pinpoint the distribution of compact regions. This distribution, in turn, is used to predict boundaries of domains in the protein. The technique provides an objective method for the location of domains both on a contact map derived from a known three-dimensional protein structure, the real distance map (RDM), and on an ADM. While most other published methods for the identification of domains locate them in the known three-dimensional structure of a protein, the technique presented here also permits the prediction of domains in proteins of unknown spatial structure, as the construction of the ADM for a given protein requires knowledge of only its amino acid sequence.     

The geometry of domain combination in proteins.

Most proteins in genomes are the result of the recombination of two or more domains. It has been found that if proteins are formed by a combination of domains from superfamilies A and B, then the domains may occur in the sequential order AB or BA but only in about 2% of cases do they occur in both sequential orders. The classical Rossmann domains of known structure are combined with catalytic domains from seven different superfamilies. In addition, there are eight cases where structures with both AB and BA domain combinations are known. For these two sets of structures, we analysed: (i) the relative orientation of the domains; (ii) the type of domain connection; (iii) the structure of the interdomain links; and (iv) domain function. The results of this analysis indicate that in most cases domain order is conserved because recombination of the domains has only occurred once during the course of evolution. Functional reasons become important when the domain connections are short. In seven out of the eight known cases where domains are combined in the AB and BA sequential orders they have different geometrical relationships that give them different functional properties.     

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.