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J Klose

Publications and source records attributed to J Klose.

66 records · Page 4Linked to original sources
PubMed1975

Gel isoelectric focusing of mouse lactate dehydrogenase: heterogeneity of the isoenzymes A4 and X4.

LDH of mouse organs (including testis) was investigated by isoelectric focusing in polyacrylamide gels. The number of LDH bands in this pattern considerably exceeds the five (six in testis) of the standard electrophoretic pattern. An attempt was made to identify these bands as tetrameric isoenzymes formed by random association of different subunits. This included isoelectric focusing of purified LDH A, B, and X, two-dimensional separation of LDH, urea treatment of LDH, staining with specific substrates, and comparison of different organs. Further experiments were performed to exclude artifacts possibly produced by the isoelectric focusing technique. Different strains of mice were also investigated. The results demonstrate that in addition to the common five LDH bands (A4-B4) one set of five bands is formed by LDH A (A41-A42) and another one by LDH X (X 41-X42). Moreover, an unusual band was found which has a lower molecular weight and no affinity to the other isoenzymes. The data suggest that the heterogeneity of the LDH pattern revealed by isoelectric focusing arises from post-transcriptional events rather than from a number of additional genes.

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Technology development at the interface of proteome research and genomics: mapping nonpolymorphic proteins on the physical map of mouse chromosomes.

Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases. The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium. Mapping of I.M.A.G.E. clones is performed in two steps: first, cDNA clones are hybridized against a 10-hit genomic mouse bacterial artificial chromosome (BAC) library. Second, interspersed repetitive sequence polymerase chain reaction (IRS-PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs. As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS-PCR fragment, the majority of cDNAs produce at least a single IRS-PCR fragment. Individual IRS fragments are hybridized against high-density spotted filter grids containing the three-dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome. IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions. This technology therefore is able to draw links between proteins detected by 2-D gel electrophoresis and the corresponding gene loci in the mouse genome.

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Genotypes and phenotypes.

Within the framework of a pilot project on the analysis of the mouse proteome, we investigated C57BL/6 mice (Mus musculus), a standard inbred strain of the mouse, starting with the analysis of brain, liver and heart proteins. Tissue extraction and the separation of proteins were performed with techniques offering a maximum of resolution. Proteins separated were analyzed by mass spectrometry. Gene-protein identification was performed by genetic analyses using the European Collaborative Interspecific Backcross (EUCIB), established from the two mouse species Mus musculus and Mus spretus. On the basis of protein polymorphisms we mapped hundreds of genes on the mouse chromosomes, allowing us new insight into the relationship between genotype and phenotype of proteins. In particular, the results showed that protein modifications can be genetically determined, therefore representing their own class of protein phenotypes. In this context, results are discussed suggesting that phenotypes of single protein species may result from several genes. Accordingly, proteins are considered as polygenic traits. In contrast, one example demonstrates that proteins may also have pleiotropic effects: a single gene mutation (a single altered protein) may affect several other proteins. From these studies we conclude that gene-related functional proteomics will show in the future that genetic diseases, defined today by clinical symptoms and considered as etiological entireties, can be subdivided into different diseases according to different affected genes.

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