Recombination between Chromosomal Is200 Elements Supports Frequent Duplication Formation in Salmonella Typhimurium

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.     

Balanced macromolecular biosynthesis in "protoplasts" of Streptococcus faecalis

Osmotically fragile forms of Streptococcus faecalis 9790 were grown in 0.5 m sucrose- or 0.5 m NH(4)Cl-stabilized medium. The "protoplast" cultures exhibit an average growth rate constant of 0.66 to 0.94 mass doublings/hr. In a variety of experiments, turbidity and the net content of protein, ribonucleic acid (RNA) and deoxyribonucleic acid increase at the same rate, indicating balanced macromolecular biosynthesis. A total of two to three mass doublings was obtained, with no evidence of cell division. After osmotic shock, "protoplast" cultures released 93 to 94% of their RNA content in a form not sedimentable at 12,800 x g for 15 min, in contrast to streptococci, which released 7% of their RNA content after the same treatment.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.