Lack Of Complementation Research Articles

Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis.

Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a "universal parent" cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

DNA-binding proteins of chick embryo lethal orphan virus: lack of complementation between early proteins of avian and human adenoviruses.

Chick cells infected by chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contained four prominent virus-specific, structurally related DNA-binding proteins with mol. wt. of 74K, 64K, 56K, 52K, and two minor forms. The CELO virus DNA-binding proteins were phosphorylated, delayed-early nuclear proteins. CELO virus early proteins were expressed in BHK cells, but did not complement human adenovirus type 5 mutants with lesions E1A, E2A or E2B. Moreover, CELO virus DNA-binding proteins were not produced in 293 cells, which express human adenovirus E1 genes. These results suggest that activation of transcription by adenovirus E1A genes involves specific interactions between the E1A gene products and viral early promoters.

Requirement of the human chromosome 11 long arm for replication of herpes simplex virus type 1 in nonpermissive Chinese hamster x human diploid fibroblast hybrids.

Somatic cell hybrids between Chinese hamster (CH) lung cells (V79/380-6), nonpermissive for productive infection by herpes simplex type 1 (HSV-1), and permissive human diploid cells support productive HSV-1 infection as long as they retain human chromosome 11. Human chromosome 3 has been reported to complement nonpermissivity in (CH) Don cells (1). Intraspecies hybrids between Don/a3 and V79/380-6 cells, however, did not support HSV-1 replication, indicating lack of complementation. The block in both nonpermissive CH cell lines was determined to involve a step beyond replication of the parental viral DNA. In cell hybrids between nonpermissive Don/a23 cells and human fibroblasts containing a t(11;15) (p11;p12) translocation, HSV-1 production was dependent solely on the presence of either human chromosome 11 or the der(11) (p11 leads to qter) translocation product containing the long arm of chromosome 11. Chromosome 3 was excluded by a discordancy rate of 59%. We conclude that the long arm of human chromosome 11 carries one or more genes coding for host functions necessary for the production of progeny HSV-1 DNA.

Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse.

We have established a cell line from mouse kidney cells expressing the tfm mutation and showed that these cells lack androgen binding activity. A subclone of these simian virus 40 (SV40)-transformed cells (6TGR-SV-tfm) selected in 6-thioguanine and lacking hypoxanthine phosphoribosyltransferase was used to produce a series of mouse--human hybrids containing the normal human X chromosome or various X autosome-translocation chromosomes (expressing only segments of the human X chromosome). When the androgen receptor locus (AR) was present in the hybrid, the number of receptor sites and kinetics of binding were similar to that in the human parental cells. Analysis of hybrids with partial human X chromosomes by using assays for X chromosome-linked enzymes and for the androgen receptor protein indicate that the AR locus on the human X chromosome is near the centromere between Xq13 and Xp11 and is proximal to the locus for phosphoglycerate kinase. Hybrids derived from 6TGR-SV-tfm mouse cells and human labial fibroblasts from an XY individual with the ar- form of androgen insensitivity have no binding activity. The lack of complementation indicates that the X chromosome-linked mutations in mouse and man affect homologous loci and supports the evolutionary conservation of X chromosomal loci in mammals; however, the position of the locus on the human X chromosome indicates that intrachromosomal rearrangement has occurred.

Complementation in vitro between Purified Mutant Fatty Acid Synthetase Complexes of Yeast

1 By dissociation and subsequent reassociation of appropriate pairs of mutant fatty acid synthetases, hybrid multienzyme complexes were obtained whose overall fatty acid synthetase activities were restored to a considerable extent. The complementation thus achieved in vitro could be both intragenic and intergenic and was, in all cases studied, in agreement with the known complementation characteristics of fatty acid synthetase (fas) mutants in vivo. 2 Similarly, the method of reversible dissociation could be used to reactivate a wild-type fatty acid synthetase which had undergone considerable loss of activity due to prolonged storage. It is believed that only those subunits which have retained their native conformation are used in the reassociation of the complex. 3 Specific component enzyme activities were determined with a variety of different mutant fatty acid synthetases and with several hybrid enzymes obtained from them by complementation in vivo or in vitro. The results indicate that the activities of most fatty acid synthetase functional domains are influenced by homologous and/or heterologous subunit interactions within the α6β6 oligomeric complex. Thus. mutational inactivation of any one of the active sites in subunit β simultaneously also alters the other four catalytic sites of this subunit. The lack of complementation occasionally observed between certain mutants of two different fas 1 complementation groups may be explained by this effect. Furthermore, α/β interactions are indicated by the fact that enoylreductase-deficient mutant enzymes (β defect) always exhibit dramatically increased β-ketoacyl synthase (α domain) activities. 4 The incorporation of FMN in vitro into wild-type fatty acid synthetase apo-enzyme from which the flavin had been removed leads to a fully active enzyme complex only when the cofactor is present during the subunit assembly process. However, addition of FMN to a fully associated apo-enzyme restores only 50% of its original overall specific activity. If FMN was replaced by FAD in either of these experiments, reactivation occurred only to an extent of 50–70% of that observed with FMN.

1028 TEMPERATURE SENSITIVITY OF ECHOVIRUS TYPE 11 CAUSING CHRONIC MENINGITIS IN AGAMMAGLOBULINEMIA

Temperature sensitive (ts) mutant viruses are associated with persistent infection (PI) of cells in vitro. An example of viral PI in human disease is chronic echovirus meningitis in agammaglobulinemic patients. We investigated temperature sensitivity in a strain of echovirus type 11 (ECHO 11) causing such illness. An isolate obtained early in the patient's course was ts, showing depressed replication at 40°C compared with 37°C (yield ratio, 40°C/37°C=10-1-10-3). Surprisingly strains of ECHO 11 that had not been associated with PI were even more ts (yield ratios 40°C/37°C=10-4-10-6). Temperature shift experiments showed that the ts step occurred late in the growth cycle of all the virus strains. Cells doubly infected with different combinations of the virus strains failed to show enhanced replication at 40°C (lack of complementation). These results suggest that all the tested strains of ECHO 11 have a ts lesion in the same gene but that in the persistently infecting strain, the lesion is “leaky.” An isolate obtained later in the patient's illness was more ts (yield ratio 40°C/37°C=10-5-10-6). Again no complementation with the other ts strains was shown. These studies indicate that the relationship between the temperature sensitivity of the infecting echovirus strain and the development of chronic meningitis in the agammaglobulinemic patient is more complex than suggested by in vitro models.

Properties of the ts3 mutant of polyoma virus during lytic infection

The ts3 mutant of polyoma virus is blocked at an early stage during lytic infection at the restrictive temperature, subsequent to virus adsorption, but prior to viral DNA synthesis. The mutant defect can be overcome by incubation at the permissive temperature in the absence of DNA synthesis prior to a shift to the restrictive temperature. Temperature sensitivity of the mutant growth is different in different cells, being greatest in BALB 3T3 cells. The growth of ts3 is not temperature-sensitive when infection is carried out with viral DNA. Complementation between ts3 and other temperature-sensitive mutants is not observed following mixed infection of BALB 3T3 cells at the restrictive temperature. This lack of complementation seems not to be caused by a dominant lethal effect of the mutant. Stimulation of resting cultures of infected BALB 3T3 cells by the addition of fresh medium and serum decreases the temperature sensitivity of the mutant growth, but only to a limited extent.

Partial dominance of lambda cIIa mutants.

A special class of lambda cII mutants, calledcIIa mutants, had previously been reported not to complement λcIII mutants. In this communication it is shown that this apparent lack of complementation is caused by a partial dominance of λcIIa mutants in co-infections with λcII+ strains. The existence of λcIIa mutants therefore cannot be taken as evidence for an oligomeric complex between the λcII and λ cIII proteins.

An S13 capsid mutant that makes no replicative-form DNA

A temperature-sensitive mutant of phage S13 ( t1069) has been isolated that fails to complement mutants in six genes tested. The reversion frequency of t1069 indicates that it is a single mutation rather than a sextuple one. The parental single-stranded DNA of t1069 is not converted to the double stranded replicative-form DNA (RF) at restrictive temperatures. Since the RF is needed for most if not all of the phage protein synthesis, the lack of complementation is explained. Heat stability tests have shown that t1069 has an altered capsid protein, and genetic crosses have located the mutant in gene IIIb. Thus at restrictive temperatures the altered IIIb capsid protein of t1069 blocks the initial steps of the normal infectious process that either precede or coincide with the replication of parental single-stranded DNA to RF.

Lysis of gram-negative bacteria by serum.

The antibacterial property of serum, first reported by Nuttall in 1888, has attracted many investigators and an extensive literature has accumulated. The sera of most mammals tested have been found to possess bactericidal activity, but a notable exception is the mouse, whose serum, apparently through lack of complement, is unable to kill any of a number of bacterial strains which are sensitive to other sera (Marcus, Esplin & Donaldson, 1954). Many types of bacteria are sensitive to serum killing, although definite lysis does not invariably occur. Most workers, including ourselves, have been concerned primarily with killing, as measured by failure to produce colonies on a culture plate. Table 1. Inactivation of the bactericidal power of human serum by inactivation of the components of complement