Colony Specificity in the Colonial Tunicate Botryllus and the Origins of Vertebrate Immunity

SYNOPSIS. Colonies of the compound tunicate Botryllus show thecapacity for self—nonself discrimination by fusion betweenseparated pieces of the same colony and rejection between piecesof unrelated colonies. We have found that genes controllingthis colony specificity are similar to those which cause transplantrejection in the vertebrates. Like the loci within the vertebratemajor histocompatibility complex (MHC), Botryllus fusibility(or histocompatibility) genes are highly polymorphic. In Botryllus,the histocompatibility complex also controls self—sterility,and limits cross—fertilization between colonies sharinghistocompatibility alleles. The mouse MHC, the H-2 region, islinked to loci which also affect the frequencies of allelesat H-2 loci in mouse populations. Thus both systems containcharacters which could act to promote the heterozygous conditionat the linked histocompatibility loci. We suggest that suchlinked characters are responsible for the evolution of allogeneicpolymorphism in vertebrates (however currently maintained),and that tunicate fusibility loci may be the evolutionary precursorsof vertebrate MHC genes.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

Evolutionary Origins of C-Terminal (GPP)n 3-Hydroxyproline Formation in Vertebrate Tendon Collagen

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)_n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)_n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)_n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)_n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)_n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.     

The Enzyme Droplets of Vertebrate Kidney

The properties and distribution of enzyme droplets of kidney, liver, and gut of several vertebrates were investigated. Qualitative and quantitative differences in enzyme content existed in droplets from different sources. There was usually a closer correlation between the properties of the inclusions and their tissue origin than between the droplets of different organs obtained from one animal. The most noticeable specific characteristic was the relatively low acid phosphatase content of the lizard tissues. In all animals the enzyme droplets of liver and gut were considerably smaller than those of the proximal region of the nephron. The collecting ducts of bird and lizard kidneys possessed droplets of a different composition from those of the proximal segment, these bodies were considered to be associated with external secretion rather than with renal function.     

The Origin of the Vertebrate Eye

In his considerations of “organs of extreme perfection,” Charles Darwin described the evidence that would be necessary to support the evolutionary origin of the eye, namely, demonstration of the existence of “numerous gradations” from the most primitive eye to the most perfect one, where each such tiny change had provided a survival advantage (however slight) to the organism possessing the subtly altered form. In this paper, we discuss evidence indicating that the vertebrate eye did indeed evolve through numerous subtle changes. The great majority of the gradual transitions that did occur have not been preserved to the present time, either in the fossil record or in extant species; yet clear evidence of their occurrence remains. We discuss the remarkable “eye” of the hagfish, which has features intermediate between a simple light detector and an image-forming camera-like eye and which may represent a step in the evolution of our eye that can now be studied by modern methods. We also describe the important clues to the evolutionary origin of the vertebrate eye that can be found by studying the embryological development of our own eye, by examining the molecular genetic record preserved in our own genes and in the genes of other vertebrates, and through consideration of the imperfections (or evolutionary “scars”) in the construction of our eye. Taking these findings together, it is possible to discuss in some detail how the vertebrate eye evolved.     

The Origin and Evolution of Vertebrate Glycine Transporters

In the vertebrate central nervous system, glycinergic neurotransmission is regulated by the action of the glycine transporters 1 and 2 (GlyT1 and GlyT2)—members of the solute carrier family 6 (SLC6). Several invertebrate deuterostomes have two paralogous glycine carrier genes, with one gene in the pair having greater sequence identity and higher alignment scores with respect to GlyT1 and the other paralog showing greater similarity to GlyT2. In phylogenetic trees, GlyT2-like sequences from invertebrate deuterostomes form a monophyletic subclade with vertebrate GlyT2, while invertebrate GlyT1-like proteins constitute an outgroup to both the GlyT2-like proteins and to vertebrate GlyT1 sequences. These results are consistent with the hypothesis that vertebrate GlyT1 and GlyT2 are, respectively, derived from GlyT1- and GlyT2-like genes in invertebrate deuterostomes. This implies that the gene duplication which gave rise to these paralogs occurred prior to the origin of vertebrates. GlyT2 subsequently diverged significantly from its invertebrate orthologs (i.e., through the acquisition of a unique N-terminus) as a consequence of functional specialization, being expressed principally in the lower CNS; while GlyT1 has activity in both the lower CNS and several regions of the forebrain.     

The Pavlovian analysis of instrumental conditioning.

An account was given of the development within the Russian literature of a uniprocess formulation of classical and instrumental conditioning, known as the bidirectional conditioning hypothesis. The hypothesis purports to offer a single set of Pavlovian principles to account for both paradigms, based upon a neural model which assumes that bidirectional (forward and backward) connections are formed in both calssical and instrumental conditioning situations. In instrumental conditioning, the bidirectional connections are hypothesized to be simply more complex than those in classical conditioning, and any differences in empirical functions are presumed to lie not in difference in mechanism, but in the strength of the forward and backward connections. Although bidirectional connections are assumed to develop in instrumental conditioning, the experimental investigation of the bidirectional conditioning hypothesis has been essentially restricted to the classical conditioning operations of pairing two CSs (sensory preconditioning training), a US followed by a CS (backward conditioning training) and two USs. However, the paradigm involving the pairing of two USs, because of theoretical and analytical considerations, is the one most commonly employed by Russian investigators. The results of an initial experiment involving the pairing of two USs, and reference to the results of a more extensive investigation, leads us to tentatively question the validity of the bidirectional conditioning account of instrumental conditioning.     

The Origins and Evolution of Culture

This article outlines a deductive theory that creates a new way to think about the origins and evolution of culture. It is Darwinian in the sense that it posits that novel concepts and behavior, like novel genes, appear randomly and are subject to selection on the basis of specific criteria that are established by the properties of living things. The theory permits us to hypothesize properties of the genome that generate culture and to infer the conditions under which selection would favor the origins of culture. Theoretical deductions lead to the conclusion that the organisms that create culture actively participate in the creation of descendants who exhibit increasing cultural abilities and who generate increases in productivity and more reliable flows of resources. Culture is not something that has evolved solely and relatively recently in the hominid line of evolution. Fossil evidence suggests that culture may have existed at least 50 million years ago, and may have originated more than 200 million years ago.     

Intracellular Organization: Evolutionary Origins and Possible Consequences to Metabolic Rate Control in Vertebrates

We adopt the position that metabolism originated at (or near)mineral surfaces prior to the origin of the first cells. Basedon current views of the organization of contemporary animalcells we speculate that the metabolism of the immediate ancestorsof eukaryotic cells required these non-biological surfaces,but that the latter were subsequently replaced by membranes,and nuclear and cytoplasmic matrix proteins which, we argue,remain as required participants in the intermediary metabolismof contemporary eukaryotic cells. The idea that such an lntracellularorganization could have provided a fundamental means by whichto control metabolic rate at the level of the intact animalis considered next. In the case of vertebrates we suggest thatthe organismic level of control might operate throughthe rateof capillary blood flow, as proposed in the Flow Theory of Coulson(1986): by controlling the rate at which the organized enzymearrays within the cells are perfused with substrate, cellularmetabolic rates could be set throughout the organism in an integratedfashion. Although there are problems with this linkage the interestingpossibility arises that the metabolic rate of individual cellsmay be subservient to the organism, being driven not so muchby the well known intracellular controls of concentration-based-biochemistryas by the flow of nutrients through the cells.     

The Molecular Evolution of the Vertebrate Trypsinogens

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted. Received: 12 July 1997