Human germline gene therapy reconsidered.

This paper reevaluates the notion of human germline gene therapy (HGLGT) in light of developments in biomedicine, biotechnology, and ethical and policy analysis. The essay makes the following key points. First, because the distinction among "therapy," "prevention," and "enhancement" is not clear in human genetics, "gene therapy" is an inadequate descriptor of the process and goals of germline genetic alterations. The alternate use of the phrase "human germline genome modification" (HGLGM) could avoid a misleading label. Second, procedures that could be construed as genetic "enhancement" may not be as morally problematic as some have supposed, once one understands that the boundaries between therapy, prevention, and enhancement are not obvious in genetic medicine. Third, HGLGM might be the medically and morally most appropriate way of avoiding the birth of a child with a genetic disease in only a small range of cases. Fourth, there are still many ethical and scientific problems relating to the safety and efficacy of HGLGM.

Sequence and genomic organization of the hsp70 genes of Leishmania amazonensis.

The sequence and genomic organization of hsp70 genes in Leishmania amazonensis were examined. Maps of overlapping cosmid clones revealed that seven L. amazonensis hsp70 genes are organized into a 24-kb locus containing 3.5-kb tandem repeats. Cosmids covering a different chromosomal region indicated that an eighth hsp70 sequence is located at a distant site. Southern blot data suggested the existence of additional hsp70 genes or pseudogenes. One complete 3.5-kb genomic repeat unit, including coding and intergenic regions, was sequenced. The predicted L. amazonensis HSP70 protein had approximately 95% sequence identity with Leishmania donovani or Leishmania major HSP70, 81-85% identity with trypanosome HSP70, and 68 or 72% identity with human HSP70 or HSP70 cognate, respectively. The GGMP tetrapeptide repeat found in other trypanosomatid HSP70 proteins is absent from the L. amazonensis sequence. Intergenic sequences of L. amazonensis and L. major differed mainly in the presence of short gaps in the L. amazonensis sequence. Potential regulatory heat shock elements were identified in the upstream sequence. Several cDNA clones were also isolated, and two different poly(A) addition sites 100 nucleotides apart were identified.

Sequence heterogeneity and polymorphic gene arrangements of the Leishmania guyanensis gp63 genes.

The Leishmania GP63 major surface protein gene family encodes multiple isoforms which differ predominantly in the carboxyterminal region. We have isolated 4 full-length gp63 cDNA clones derived from stationary-phase promastigote RNA of a cloned isolate of Leishmania guyanensis, a member of the braziliensis complex. These genes, along with the previously published L. guyanensis gp63 gene sequence [15], appeared to be mosaics of different combinations of 5' and 3' untranslated regions and sequences encoding the propeptide, internal, and C-terminal regions of GP63. The predicted L. guyanensis GP63 isoforms shared as little as 55% sequence identity, comparable to the inter-species diversity of GP63. The genomic organization of gp63 genes in L. guyanensis is highly complex: there are at least 4 distinct polymorphic forms of tandemly linked gene clusters, with intra-gene cluster variation in gene sequence and in the number of gene repeats. Southern blot analysis suggested that the arrangement of gp63 genes in this L. guyanensis isolate did not differ from that in the parental lines.

Sequence of a Leishmania major gene encoding an ubiquitin fusion protein.

A gene encoding an ubiquitin-tail protein fusion was isolated from the parasitic protozoan, Leishmania major, and sequenced. The L. major tail protein shares 97, 96, 67, 62, 62 and 61% sequence identity with the tail proteins of Trypanosoma brucei, Trypanosoma cruzi, yeast, Dictyostelium discoideum, human, and Arabidopsis thaliana, respectively. The putative 'zinc finger' nucleic acid-binding domain found in all ubiquitin 'tail' or 'extension' proteins described is also conserved in the L. major sequence. The upstream sequence indicated that this gene is not located at the end of a polyubiquitin sequence.

Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and beta-tubulin in cloned strains of several Leishmania species.

The molecular karyotypes of several Leishmania isolates (Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 major surface glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and beta-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The beta-tubulin gene probe hybridized predominantly to large (1600-1750 kb) chromosome-size DNA and to 1-5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica. Gp46/M2 genes were located on 1 band in L. donovani, L. major, and L. aethiopica or 2 bands in L. tropica and L. amazonensis, but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii. Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.

Molecular cloning and characterization of the immunologically protective surface glycoprotein GP46/M-2 of Leishmania amazonensis.

Immunization of mice with the GP46/M-2 membrane glycoprotein has been demonstrated to elicit protection against infection with the parasitic protozoan Leishmania amazonensis. As this molecule is important for future vaccine studies of leishmaniasis, the gene encoding the GP46/M-2 surface membrane glycoprotein of Leishmania amazonensis has been cloned and sequenced. The protein sequence derived from the DNA sequence data is consistent with the known biochemical and immunochemical properties of the protein and indicates a number of structural areas of interest. A repetitive sequence (24 amino acids repeated four times) occurs within the amino-terminal portion of the molecule and constitutes approximately 22% of the total mature protein. The protease-resistant immunodominant carboxyl-terminal domain of the protein comprises approximately half of the molecule and consists of proline-rich and cysteine-rich areas of sequence; the distribution of cysteine residues is suggestive of metal binding motifs. The sequence predicts a hydrophobic leader peptide, and a putative attachment site for a glycosyl-phosphatidylinositol anchor is indicated at the carboxyl terminus, consistent with the membrane location of the protein. Southern blot analyses also indicate the presence of a GP46/M-2 gene family.

Flagellar membrane and paraxial rod proteins of Leishmania: characterization employing monoclonal antibodies.

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis.

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA+ strain but more than pKM101 in a uvrA- strain. muc- point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.

Mutagenesis and cellular responses to DNA damage.

Treatment of Escherichia coli with DNA-damaging agents results in the increased expression of a set of din (damage-inducible) genes. We have studied the regulation and function of these genes by using the Mud(Ap, lac) bacteriophage to obtain fusions of the beta-galactosidase structural gene to the promoters of various din genes. By this technique, we have shown that the uvrA, uvrB, and umuC genes are induced by UV and other DNA damaging agents. The products of the uvrA and uvrB genes are required for the excision repair of pyrimidine dimers and other bulky lesions; the umuC gene product is required for most chemical mutagenesis in E. coli. Genetic analyses of all of the din-lac fusions isolated to date indicate that lexA is the direct repressor of each of the din genes and that proteolytic cleavage of the lexA protein is required for their induction. We have also been studying the mechanism by which the clinically isolated plasmid pKM101 increases the susceptibility of cells to chemical mutagenesis. Inasmuch as the effects of pKM101 on mutagenesis are recA+ lexA+ -dependent and the plasmid can suppress the nonmutability of a umuC mutant, it seems likely that pKM101 may carry an analog of the chromosomal umuC gene. By insertion mutagenesis using Tn5, we identified an approximately 2,000-base pair region of pKM101 which is necessary for its effects on mutagenesis.

Functional organization of plasmid pKM101.

Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.

Restriction endonuclease cleavage map of pKM101: relationship to parental plasmid R46.

A detailed restriction endonuclease cleavage map of the plasmid pKM101 has been constructed. pKM101 plasmids containing individual Tn5 insertions were used to facilitate the ordering of restriction fragments generated by enzymes cleaving pKM101 at multiple sites. By restriction enzyme analysis, pKM101 (35.4 kilobases) appears to have arisen from its clinically-isolated parent by deletion of a single DNA region which codes for three of the four drug resistances carried by R46.

Asynchronous synthesis of erythrocyte membrane proteins.

The synthesis of membrane proteins of the mature mouse erythrocyte is asynchronous. During erythropoiesis, synthesis of the bulk of the spectrin and actin polypeptides is completed before that of the major transmembrane glycoprotein. Synthesis of the glycoprotein ceases before that of several minor proteins found on the inner surface of the red cell membrane, and one of these minor proteins is made predominantly by reticulocytes. These findings were the result of experiments in which a normal mouse was given a single injection of [35S]methionine. The appearance of radioactivity in the membrane proteins of circulating mature erythrocytes was followed. The earliest labeled proteins to emerge into the blood represent those synthesized at the last stages of erythropoiesis.