Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

Sequencing Studies of Icr-170 Mutagenic Specificity in the am (Nadp-Specific Glutamate Dehydrogenase) Gene of NEUROSPORA CRASSA

The acridine half-mustard ICR-170-induced reversion of the mutant am15, which has a single base-pair deletion, at a frequency of between 9 and 28 X 10(-6). In each of three classes of revertants, the mutagen had induced the insertion of a -G- -C- base pair at a -G-G- -C-C- site. The mutant am6, which has a single base pair insertion, is known to be revertible, with UV light, by deletion of a -G- -C- base pair at a -G-G-G- -C-C-C- site. This mutant reverted with ICR-170 at a frequency of 0.1 X 10(-6). These results show that ICR-170 is able to induce addition frameshifts in Neurospora crassa within short, monotonous runs of G:C base pairs, but indicate a lack of deletion activity at such sequences.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens

Summary The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present.The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens.In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens.The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.