Gunther S. Stent

Phage and the origins of molecular biology

An expanded edition of the acclaimed collection of 35 essays by pioneers of microbiology, first published in 1966 as a 60th-birthday tribute to Max Delbruck.

Neuronal control of swimming in the medicinal leech

SummaryLeeches swim by undulating their extended and flattened body in the dorsoventral direction, to form a wave that travels backwards along the animal. The troughs and crests of this body wave are produced by a metachronal rhythm of antiphasic contractions of the dorsal and ventral longitudinal musculature of the body wall of successive segments. Cinematographic records of swimming leeches show that over a range of cycle periods from 390 to 1100 msec the animal maintains one full wave along the length of its body. This constant wave form is achieved by compensating for the increase in the cycle period by an increase in the intersegmental travel time of the wave from 19 to 77 msec per segment. Direct muscle tension measurements of the segmental body wall during the swimming movement of a restrained, brainless and partially denervated leech lead to values for the dynamic parameters of the body wave which are in agreement with those abstracted from cinematographic records. In order to study the neuronal control of the swimming movement, a semi-intact leech preparation was developed. This preparation carries out movements that are clearly vestiges of the swimming rhythm while allowing the taking of electrophysiological records from exposed and immobilized parts of its peripheral and central nervous systems. The records reveal a nerve cell activity rhythm whose period matches that of the swimming rhythm. The swimming rhythm and its body wave must be generated by a system of distributed, phase-locked segmental oscillators that cause an antiphasic activity of the motor neurons innervating the segmental dorsal and ventral longitudinal muscles. The cycle of these oscillators can be inferred to consist of a variable time sector whose changes in length are responsible for changes in the cycle period, and of a constant time sector whose length is independent of the cycle period.

Direction of chain growth in enzymic RNA synthesis

The terminal nucleotides of poly-A and RNA synthesized enzymically in vitro were identified by eleetrophoretie analysis of alkaline hydrolysates of the polymers. It was ascertained which terminus represents the starting and which the growing end of the polynucleotide chains, by changing the specific activity of the radioactive substrate during the polymerization reaction. The following results were obtained. (1) In alkaline hydrolysates of both poly-A and RNA the 3′-terminal nucleotide appears as a nucleoside, and the 5′-terminal nucleotide as the nucleoside tetraphosphate pppXp. (2) RNA synthesis proceeds from the 5′-to the 3′-end of the molecule. (3) RNA chains are initiated predominantly by pppA. (4) Throughout the first 60 minutes of poly-A synthesis, growth of new chains is initiated and growth of old chains is terminated, the average poly-A molecule growing for ten minutes at a rate of 0·6 nucleotide/sec to an ultimate length of 360 nucleotides. (5) In RNA synthesis, growth of most chains is initiated during the first ten minutes of reaction. Growth of about half of the chains stops after a few minutes, whereas growth of the remainder continues for a much longer period at a rate, depending on the substrate concentration, from 0·7 to 2·5 nucleotides/sec/chain.

Embryonic origins of cells in the leech Helobdella triserialis

To ascertain the embryonic origins of the cells in various tissues of the leech Helobdella triserialis, horseradish peroxidase (HRP) was injected as a cell lineage tracer into all identified blastomeres of the early embryo in turn, except for a few of the micromeres, and the resulting distribution of HRP-labeled cells was then examined in the late embryo. In this way it was found that in every body segment a topographically characteristic set of neurons in the ganglion and body wall and a characteristic territory of the epidermis is derived from each of the four paired ectodermal teloblasts N, O/P, O/P, and Q, whereas the muscles, nephridia, and connective tissue, as well as a few presumptive neurons in each segmental ganglion, are derived from the paired mesodermal teloblast, M. Each topographically characteristic, segmentally iterated set of neurons descended from a given teloblast is designated as a kinship group. However, the prostomial (nonsegmental) epidermis and the neurons of the supraesophageal ganglion were found to be derived from the a, b, c, and d micromere quartet to which the A, B, C, and D blastomeres give rise at the dorsal pole of the embryo. The superficial epithelium of the provisional integument, which covers the surface of the embryo midway through development and is sloughed off at the time of body closure, was found to be derived from the a, b, c, and d micromere quartet, as well as from other micromeres produced in the course of teloblast formation. The contractile fibers of the provisional integument were found to be derived from the paired M teloblast. These results demonstrate that development of the leech embryo proceeds according to a highly stereotyped pattern, in the sense that a particular identifiable blastomere of the early embryo regularly gives rise to a particular set of cells of the adult (or provisional embryonic) tissues.

Mating in the reproduction of bacterial viruses.

Publisher Summary This chapter focuses on some special aspects of bacteriophage growth. It deals with the question of whether a genetic interaction may occur between bacteriophage and hostcell genome not only during the lysogenic cycle but also during the Zytic cycle. The chapter considers the lethal effects produced in bacterial viruses and their host cells by the decay of incorporated P32 atoms and by X- and UV irradiation. It was seen that a variety of bacterial viruses can be divided into two groups. The chapter attempts to show that the differences between these two groups can be understood on the basis of a unitary hypothesis: the phages of the second group mate repeatedly with the genome of the host cell during their intracellular reproduction, whereas those of the first group do not. The chapter also proposes a molecular mechanism of replication of DNA, which was designed principally to incorporate the notion that the mating of hereditary structures of the vegetative phages is a necessary aspect of their replication.

Generation of a locomotory rhythm by a neural network with recurrent cyclic inhibition

An oscillatory intersegmental neuronal network drives the swimming rhythm of the leech. This network consists of interneurons joined via inhibitory connections to form a series of segmentally iterated, concatenated rings. Recurrent cyclic inhibition in these rings produces a multiphasic activity rhythm. By theoretical analysis of such concatenated interneuronal rings and construction of their electronic analogs it is shown that the interneural network identified in the central nervous system of the leech has properties appropriate for generating the observed motor output.

Embryonic cell lineages in the nervous system of the Glossiphoniid leech Helobdella triserialis

Abstract The lines of descent of cells of the nervous system of the leech Helobdella triserialis have been ascertained by injection of horseradish peroxidase (HRP) as a tracer into identified cells of early embryos. Such experiments show that the nervous system of the leech has several discrete embryological origins. Some of the neurons on one side of each of the segmental ganglia derive from a single cell, the ipsilateral N ectoteloblast. Other neurons derive from a different precursor cell, the ipsilateral OPQ cell that gives rise to the O, P, and Q ectoteloblasts. The positions within the ganglion of neuronal populations derived from each of these sources are relatively invariant from segment to segment and from specimen to specimen. Other nerve cord cells derive from the mesoteloblast M; of these four per segment appear to be the precursors of the muscle cells of the connective. The A, B, or C macromeres contribute cells to the supraesophageal ganglion. In preparations in which an N ectoteloblast was injected with HRP after production of its bandlet of n stem cells had begun, the boundary between unstained (rostral) and stained (caudal) tissues can fall within a ganglion or between ganglia. This suggests that each hemiganglion contains the descendants of more than one, and probably two, n stem cells.

RNA chain growth rates in Escherichia coli

Abstract The rate of RNA chain growth was measured in vivo in Escherichia coli cultures growing in various media at 29 and 37 °C. For this purpose, the bacteria were allowed to assimilate [ 3 H]uracil or [ 3 H]guanine into their RNA for short time-periods. The RNA was then extracted and hydrolyzed with alkali, and the radioactivity measured in the resulting nucleotides and nucleosides. The data thus obtained allowed the calculation of individual nucleotide step-times in RNA chain growth, the step-time for a particular nucleotide being defined as the average time required for adding the next nucleotide to the end of a nascent RNA chain carrying that particular nucleotide as its growing end. In bacteria growing exponentially in a glucose-Casaminoacids medium at 29 °C with 1.07 cell generations per hour, the uridylic acid step-time was estimated to be 42 msec, the cytidylic acid step-time to be at most 67 msec and the guanylic acid step-time to be 20 msec. Assuming an equality of the step-times of guanylic and adenylic acids, the average RNA chain growth rate was estimated to be 26 nucleotides/second at 29 °C. At 37.5 °C both the generation period as well as the nucleotide step-times were found to be 60% of the corresponding values at 29 °C, giving an average RNA chain growth rate of 43 nucleotides/second. The nucleotide step-times in E. coli growing at 29 °C in a succinate medium with 0.63 cell generation per hour and in a proline medium with 0.33 cell generation per hour were found to be longer than the corresponding step-times during the faster growth in glucose-Casaminoacids medium. The increases in nucleotide step-times with increases in the generation period observed here are not nearly great enough, however, to account for the great variations in the rate of RNA synthesis observed under these three different physiological conditions. Thus bacteria appear to adjust their specific content of RNA according to their physiological needs by varying both the rate of RNA chain growth and the number of nascent RNA molecules under synthesis at any time.

Neuronal control of heartbeat in the medicinal leech

SummaryThe leech heartbeat consists of a constriction-dilation rhythm of two lateral heart tubes extending over the length of the body. The beats of the segmental sections of these two tubes are coordinated in such a manner that the heart tube of one body side produces a frontward peristaltic wave while the heart tube on the other body side produces nearly concerted constrictions. This rhythm is metastable, in that left and right heart tubes alternate between peristaltic and concerted constriction modes, with a given mode lasting for tens or hundreds of beat cycles.The constriction-dilation cycles of the segmental heart tube sections are controlled by a set of rhythmically active motor neurons, the heart excitors, or HE cells. A bilateral pair of HE cells is located in all but the two frontmost and the two rearmost segmental ganglia of the ventral nerve cord. Each HE cell innervates via excitatory synapses the circular muscle fibers in the wall of the ipsilateral heart tube section. The activity cycle of the HE cells consists of an active phase, during which they are depolarized and produce a burst of impulses, and an inactive phase during which they are repolarized by a burst of inhibitory synaptic potentials. The intersegmentally coordinated activity cycles of the HE cell set are maintained in an isolated ventral nerve cord. Hence the generation of the heart excitor rhythm does not require sensory feedback.

Cell lineage in the development of the leech nervous system

The intricate structure and function of the adult nervous system is the result of developmental interactions of factors both intrinsic and extrinsic to the embryonic neurons and their precursor cells. To fathom the mechanisms underlying these interactive processes, a detailed knowledge of the course of neurogenesis at the cellular level is essential. Once such knowledge is available, specific and well-focused questions can be formulated at the biophysical, biochemical, or genetic levels. One key aspect of the process of neurogenesis at the cellular level is cell lineage, i.e., the embryonic lines of descent of various types of neurons. The importance of cell lineage for understanding developmental processes was realized over a century ago by C. O. Whitman.1 On the basis of his studies of the development of leeches, Whitman put forward the idea, then quite novel, that each identified cell of the early embryo, and the clone of its descendant cells, plays a specific role in later development. Cell lineage analyses were later extended to the embryos of other species, not only by direct observation but also by use of other techniques, such as selective ablation, application of extracellular marker particles, and, most importantly, production of chimera and genetic mosaics.2–10 More recently, we have refined and extended Whitman’s century-old cell lineage studies in leech embryos, with particular emphasis on the cellular origins of the leech nervous system. As will be seen in this chapter, leeches are well suited for cellular investigations of neuronal development because both their early embryos and their adult nervous systems comprise identifiable cells accessible to experimental manipulation.