Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They were terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was <4°C for three of the 18 families, and <10°C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55°C in 0.18 M Na⁺. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.     

Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis.

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.     

Calculation of CO2 gas phase diffusion in leaves and its relation to stomatal resistance

A new theory and experimental method was developed to measure the diffusion resistance to CO₂ in the gas phase of mesophyll leaf tissue. Excised leaves were placed in a chamber and their net evaporation and CO₂ assimilation rates measured at two different ambient pressures. These data were used to calculate CO₂ gas phase diffusion resistances. A variety of field grown leaves were tested and the effects of various experimental errors considered. Increasing the gas phase diffusion resistance decreased transpiration more than it decreased CO₂ assimilation. It was concluded that gas phase diffusion resistance associated with CO₂ assimilation may sometimes be 100 or 200 s·m^-1 greater than the resistance implied by transpiration rates. This may be due to longer path lengths for the CO₂ diffusion, constricted in places by the shape and arrangement of mesophyll cells.     

A pH-dependent interaction between histones H2A and H2B involving secondary and tertiary folding.

It has been shown by high-resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea-denatured acid-extracted and from urea-denatured salt-extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength. From CD spectra the H2A/H2B complex is shown to contain about 37% alpha helix but no beta structure, the latter being confirmed by infrared spectroscopy in the 6-mum region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A. The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31-95 of H2A and residues 37-114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.     

Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli.

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of Mr of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of Nuclear Thyroid Hormone Receptors in Cerebral Cortex In Vivo

Abstract: Investigation was conducted under in vivo conditions in the adult male rat to determine the basic characteristics of the nuclear thyroid hormone receptors in the cerebral cortex. Equilibrium with cortical nuclei of an intravenous dose of triiodothyronine (T₃) occurred 3 h after injection and showed a t _1/2 of 1 h for dissociation. Saturation of receptors occurred at 0.5–0.6 ng/mg DNA. The endogenous level of binding in the normal rat was 0.07–0.1 ng/mg DNA, representing a 15% occupancy of total receptors at a serum concentration of 66 ng/dl. These characteristics were then examined under several pathophysiological conditions. In the hypothyroid rat, an apparent 37% in-crease in total binding sites occurred. Under either fasting conditions or insulin or glucagon administration declines in serum T₃ were noted, and the endogenous binding also decreased in parallel. Only glucagon produced a significant reduction in total binding sites. Under the hypoxic condition produced by maintenance under a 7% oxygen atmosphere, a slight increase in apparent total binding sites was found with no change in endogenous binding level. Severe narcosis resulted in no effects on T₃ binding parameters. These results demonstrate specific alterations of thyroid hormone receptors that may be important physiologically.     

Comparison of the three‐dimensional structures of a humanized and a chimeric Fab of an anti‐γ‐interferon antibody

The objective of this work is to compare the three‐dimensional structures of “humanized” and mouse–human chimeric forms of a murine monoclonal antibody elicited against human γ‐interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen‐binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8000 by nearly identical microseeding procedures. Their structures were solved by X‐ray analyses at 2.9 Å resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity‐determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized V_H, the substitution of serine for proline in position 7 allowed the N‐terminal segment (designated strand 4‐1) to be closely juxtaposed to an adjacent strand (4‐2) and form hydrogen bonds typical of an antiparallel β‐pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an α‐helix involving residues 25–32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 Å to a random coil which terminated in a single turn of 3₁₀ helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space‐saving α‐helix, contrasting with a more extended 3₁₀ helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use. Copyright © 1999 John Wiley & Sons, Ltd.     

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.     

Virulence signatures: microarray-based approaches to discovery and analysis

Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information. Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species. Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens. By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay. We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded. We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.