Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.     

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.     

The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase

The rabbit reticulocyte Mr 90,000 protein associated with the heme-sensitive eIF-2 alpha kinase has been identified previously as the mammalian heat shock protein of this size class (hsp 90). Purified reticulocyte hsp 90 when added exogenously to the kinase increases its activity. This stimulatory effect is abolished after incubation of hsp 90 with a highly purified type 1 phosphoprotein phosphatase isolated from reticulocytes. Phosphorylation of dephosphorylated hsp 90 by casein kinase II but not by cAMP-dependent protein kinase restores the biological activity of hsp 90 to stimulate eIF-2 alpha phosphorylation.     

The valve movement response of mussels: a tool in biological monitoring

Biological sensors are becoming more important to monitor the quality of the aquatic environment. In this paper the valve movement response of freshwater (Dreissena polymorpha) and marine (Mytilus edulis) mussels is presented as a tool in monitoring studies. Examples of various methods for data storage and data treatment are presented, elucidating easier operation and lower detection limits. Several applications are mentioned, including an early warning system based on this valve movement response of mussels.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Molecular cloning of complementary DNA encoding maize nitrite reductase: molecular analysis and nitrate induction

Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.     

The role of air breathing in the resistance of bimodally respiring fish to waterborne toxins

The bimodally respiring catfish Clarias macrocephalus Günther responded to a toxic extract of Croton tiglium (Euphorbiaceae) seeds by increased air breathing under both normoxic (8.1 ± 0.4 mgO₂ l⁻¹) and hypoxic (0.7 ± 0.1 mgO₂l⁻¹) conditions. Fish in hypoxia survived longer than those in normoxia when surface access was provided. When air breathing was prevented, survival time in toxin was greatly reduced at both levels of dissolved oxygen, and fish in normoxia survived longer than those in hypoxia. Non-toxin controls without surface access survived in normoxia but in hypoxia died at the same time as the fish in toxin. These results suggest that air breathing increases the resistance offish to toxins by permitting a decrease in the rate of gill ventilation and hence the rate at which toxins are absorbed.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.