Biotherapies of neuromuscular disorders.

This review focuses on the most recent data on biotherapeutic approaches, using DNA, RNA, recombinant proteins, or cells as therapeutic tools or targets for the treatment of neuromuscular diseases. Many of these novel technologies have now reached the clinical stage and have or are about to move to the market. Others, like genome editing are still in an early stage but hold great promise.

Morphological and molecular characterization of new Drosophila cell lines established from a strain permissive for gypsy transposition.

The gypsy element of Drosophila melanogaster is the first retrovirus identified in invertebrates. Its transposition is controlled by a host gene called flamenco (flam): restrictive alleles of this gene maintain the retrovirus in a repressed state while permissive alleles allow high levels of transposition. To develop a cell system to study the gypsy element, we established four independent cell lines derived from the Drosophila strain SS, which contains a permissive allele of flamenco, and which is devoid of transposing copies of gypsy. The ultrastructural analysis of three SS cell lines revealed some remarkable characteristics, such as many nuclear virus-like particles, cytoplasmic dense particles, and massive cisternae filled with a fibrous material of unknown origin. Gypsy intragenomic distribution has been compared between the three cell lines and the original SS fly strain, and revealed in two of the cell lines an increase in copy number of a restriction fragment usually present in active gypsy elements. This multiplication seems to have occurred during the passage to the cell culture. Availability of SS cell lines should assist studies of gypsy transposition and infectivity and might be useful to produce high amounts of gypsy viral particles. These new lines already allowed us to show that the Envelope-like products of gypsy can be expressed as membrane proteins.

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.

Expression of a foreign KmR gene in linear killer DNA plasmids in yeast.

The killer plasmids of the yeast Kluyveromyces lactis, pGKL1 and 2 (k1 and k2 for short), are linear double-stranded DNAs. The expression of genes of these plasmids is thought to depend on their own transcription system. Cloning the plasmid genes in conventional circular vectors is therefore not suitable for transcriptional studies, because such vectors use the host nuclear transcription system. In vitro modification of the linear plasmid genomes in order to introduce transcription reporter genes has been difficult because the structure of the plasmids, with covalently bound terminal proteins, does not allow their manipulation in vitro and amplification in Escherichia coli. We introduced the kanamycin/G418 resistance gene, KmR, into the k1 plasmid in vivo, by transforming the yeast with the linearized KmR gene bordered with short k1 sequences (part of the region encoding the toxin) to allow homologous recombination with the resident k1. In the linear recombinants obtained, however, the KmR was not expressed, while it was expressed if carried on circularized plasmids. By replacing the native promoter of KmR by the ORF1 promoter from k1, the KmR gene could be expressed in linear recombinants and conferred on the host a high level of resistance to the drug. All the linear recombinant plasmids were extremely stable under nonselective conditions. As a rare event, the integration of KmR produced a palindromic rearrangement of the k1 plasmid.

The Kluyveromyces lactis KEX1 gene encodes a subtilisin-type serine proteinase.

KEX1 is a chromosomal gene required for the production of the killer toxin encoded by the linear DNA plasmid pGKL-1 of Kluyveromyces lactis. The nucleotide sequence of the cloned KEX1 gene has been determined. The deduced structure of the KEX1 protein, 700 amino acids long, indicated that it contained an internal domain with a striking homology to the sequences of the subtilisin-type proteinases, and a probable transmembrane domain near the carboxyl terminus. The results confirm the hypothesis that the product of the gene KEX1 of K. lactis is a proteinase involved in the processing of the toxin precursor.

A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis.

The killer system of Kluyveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which block the expression of the killer character have been isolated. These mutations reside in a single chromosomal gene which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a disrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutation of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutation of S. cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.

A gene-cloning system for Kluyveromyces lactis and isolation of a chromosomal gene required for killer toxin production.

A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyces lactis. The principle is essentially equivalent to that of the 2 microns/Saccharomyces cerevisiae transformation system. The main features of the system are presented. Using a pKD1-based DNA bank of K. lactis, the KEX1 gene involved in the killer system was isolated by complementation.