A transfection compound series based on a versatile Tris linkage.

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Multiple domains in a ribozyme construct confer increased suppressive activity in monkey cells.

An expression vector designed to express a long (approximately 1 kb) RNA containing multiple ribozyme domains was cotransfected into mammalian cells with a plasmid encoding, as target, messenger RNA for chloramphenicol acetyltransferase (CAT). In comparative studies the multimeric ribozyme construct proved to be significantly more effective at suppressing CAT expression than either the corresponding antisense RNA, or a transcript carrying a single ribozyme domain. Suppression of gene activity was apparently specific because expression of an independently expressed gene for human growth hormone was unaffected. The profile of CAT RNA extracted from transfected cells was consistent with RNA cleavage.

Inhibition of gene expression by a short sense fragment.

In order to further investigate the mechanism of antisense inhibition of gene expression, a series of plasmids which generate short antisense RNAs deriving from the 5' end of the CAT gene were constructed. When transfected into COS1 cells, these constructions were capable of specifically reducing CAT gene expression. Unexpectedly, transfection with constructions expressing defective RNA in the sense orientation also resulted in reduced levels of both CAT enzyme and mRNA. This was mediated by both short and full-length CAT-gene fragments, and was dependent on the presence of the tested transcriptional promoters, either the herpes simplex virus thymidine kinase (TK) or simian virus 40 late (SVL) promoters. These results, in conjunction with computer aided secondary structure prediction, indicate a possible similarity of regulatory mechanism for both senses of RNA acting within the cell.

Specific gene suppression by engineered ribozymes in monkey cells.

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.

Failure of Klebsiella pneumoniae antibodies to cross-react with peripheral blood mononuclear cells from patients with ankylosing spondylitis.

Cross-reactivity between antibodies to 2 strains of Klebsiella pneumoniae (K43 and F77) and the peripheral blood lymphocytes of patients with ankylosing spondylitis (AS) was examined in 3 separate antibody binding and cytotoxicity assays. Using K pneumoniae antisera in a chromium release cytotoxicity assay, we found no difference in the reactions of cells from AS patients and those from control subjects. This result contrasts with the results of previous studies. Similarly, using an enzyme-linked immunosorbent assay, we detected no significant increase in antibody binding to peripheral blood mononuclear cells (PBMC) in HLA-B27 positive patients with AS. Low levels of antibody binding were detected by a fluoresceinated antibody binding assay; however, normal rabbit serum, which was used as a control, was shown to have a binding affinity for PBMC that was significantly greater than that of specific K pneumoniae antisera. The results of our present study do not support the concept of a specific cross-reactivity between antibodies to K pneumoniae and the PBMC of patients with AS who are HLA-B27 positive.

Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388.

The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined. The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons. A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region. The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1. Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa. This segment includes a 59 base element previously found flanking the Tn7 aadA gene. A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.

Studies of macrophage function in murine systemic lupus erythematosus. 4. Failure to reverse the defect in Fc-mediated phagocytosis and binding by in vitro stimulants or prostaglandins.

Previous studies have shown that resident peritoneal macrophages from mice with systemic lupus erythematosus (SLE) show defective Fc-mediated phagocytosis and binding of opsonized sheep erythrocytes (EA) in vitro. Possible causes of this defect in (NZB X NZW)F1 (B/W) mice were investigated. These included a maturational block in peritoneal macrophage differentiation, the production by peritoneal cells of a factor which inhibits Fc receptor expression and phagocytosis, and an abnormal response by macrophages of autoimmune mice to prostaglandins. Resident peritoneal macrophages of B/W mice did not show a maturational block since incubation with either (a) differentiating agents such as 4 beta-phorbol 12 beta-myristate 13 alpha acetate or retinoic acid, or (b) lymphokine (LK), prepared by Con A stimulation of mouse spleen cells, failed to enhance Fc-mediated phagocytosis and binding by B/W cells relative to controls. However, LK from B/W and B6AF1 cells stimulated Fc-mediated phagocytosis and binding by bone-marrow (BM)-derived macrophages of CBA/H mice; B/W LK also stimulated BM cells from B/W mice. Peritoneal cell supernatants did not inhibit phagocytosis of Fc receptor expression by BM-derived macrophages in vitro. Prostaglandin E treatment of peritoneal or BM-derived macrophages in vitro failed to restore decreased phagocytosis and binding of EA induced by culture in indomethacin and failed to stimulate phagocytosis by untreated cultures. The reason for the observed defect remains obscure.

Studies of macrophage function in murine systemic lupus erythematosus. 3. The nature, anatomical location, and reversibility of the phagocytic defect.

The defect in phagocytosis and binding of antibody-coated sheep erythrocytes (EA) by peritoneal macrophages of (NZB X NZW)F1 or B/W mice is not intrinsic, but is related to the development of the autoimmune disease process. The defect appears to be confined to peritoneal macrophages, since bone marrow (BM)-derived macrophages have normal to elevated activities in vitro. The peritoneal macrophage defect is not due to blockade of Fc receptors in vivo, as shown by long-term culture or recovery of phagocytic and binding activities after removal of Fc receptors by pronase, but represents a reduced number of receptors with slightly delayed turnover. The defect can be reversed by elicitation of activated macrophages with Corynebacterium parvum, thioglycollate, or proteose peptone in vivo. Normal Fc-mediated phagocytosis and binding by BM-derived macrophages cultured from untreated autoimmune mice is enhanced by pretreatment of mice with C. parvum, thioglycollate, or proteose peptone. The cause of the defect in Fc-mediated phagocytosis by resident peritoneal macrophages of autoimmune mice was not ascertained; it may be due to abnormal macrophage kinetics or to the local effects of lymphokines released as a result of other autoimmune changes.

Construction of a gentamicin resistance gene probe for epidemiological studies.

A 7.7-kilobase BamHI fragment was cloned from the transconjugant of a clinical isolate of Escherichia coli containing a 120-kilobase multiresistance IncC plasmid. The recombinant plasmid conferred resistance to kanamycin, gentamicin, tobramycin, sulfamethoxazole, and trimethoprim. This clone was used to generate a series of subclones from which a 2.0-kilobase BamHI-HindIII probe containing a gentamicin 2''-O-adenylyltransferase [AAD(2'')] gene was obtained. This probe hybridized specifically in both colony and Southern hybridizations with the AAD(2'') gene but not with other resistance genes, including other aminoglycoside-modifying genes, or with a reference IncC plasmid lacking the AAD(2'') gene. The AAD(2'') gene may be part of a transposon, since hybridization occurred with both nonconjugative plasmids and the chromosome in some isolates.

Occurrence of chloramphenicol acetyltransferase and Tn9 among chloramphenicol-resistant enteric bacteria from humans and animals.

The occurrence of chloramphenicol acetyltransferase (CAT) among chloramphenicol resistant enteric bacteria from humans, animals (cats, dogs) and sewage was examined. The enzyme appears to be the basis of resistance in 83 and 84 per cent of bacteria of humans and sewage origin, and in 50 per cent of bacteria from animals. In order to identify type I CAT among chloramphenicol resistant isolates, total cellular DNA was probed with a 32P-labelled fragment of the CAT structural gene from the transposable element Tn9. Nineteen per cent of chloramphenicol resistant enteric bacteria of clinical origin, 11% of sewage isolates, and 11% of veterinary isolates gave positive hybridization results. The difference between bacteria of clinical and veterinary origin in respect of both parameters tested is significant and is interpreted as indicating genetic dis similarity between the two pools in regard to chloramphenicol resistance. This may reflect a lack of contact between the two pools, or host bacterial factors with select against CAT-mediated (and type I CAT more specifically) resistance to chloramphenicol.

Is a Klebsiella plasmid involved in the aetiology of Ankylosing Spondylitis in HLA-B27-positive individuals?

The possibility that plasmid genes, carried by enteric organisms previously indirectly implicated as disease agents, play a role in the pathogenesis of Ankylosing Spondylitis (AS) was explored. A particular Klebsiella isolate (K21) previously found to cross-react with cells from HLA-B27 positive (B27+) patients with AS, but not with cells from normal individuals, was found to contain a plasmid(s). This coded for the organism's ability to produce a factor which could modify B27+ normal cells (AS-) rendering them lysable by the anti-Klebsiella serum. Curing of this isolate resulted in the loss of the plasmid concerned and a loss of ability of its culture filtrate to modify B27+ lymphocytes of clinically healthy subjects. When plasmids from K21 were transferred to a plasmid free laboratory strain, E. coli JP995, the recipient strain acquired the ability to elaborate modifying factor. These data suggest that plasmids, harboured by some enteric bacteria, and their products, may be implicated in modifying cells bearing certain Major Histocompatibility Complex genes, and that such modification may be an important factor in the pathogenesis of a number of diseases including the seronegative arthropathies.