A targeted kappa immunoglobulin gene containing a deletion of the nuclear matrix association region exhibits spontaneous hyper-recombination in pre-B cells.

Previous studies employing ectopic integration of reporter genes have shown that the nuclear matrix association region (MAR) adjacent to the intronic enhancer of the mouse kappa immunoglobulin (Ig) gene is required for high level transcription of rearranged genes, demethylation, reduction of position effects and maximal somatic hypermutation in B cells. To test for the function of this MAR in its natural chromosomal environment, we pursued the 'HIT-and-RUN' procedure with the mouse pre-B cell line 103 to create a targeted MAR deletion. We observed a 'HIT' targeting frequency of 1/684 but 0/2100 'RUN' clones maintained the MAR-deleted germline locus because of an unexpected hyper-recombination for Vkappa-Jkappa joining, specifically to the MAR-deleted allele, and primarily at Jkappa4 and Jkappa5. This hyper-recombination was correlated with undermethylation of the Jkappa-Ckappa region but not with the level of local transcription. These results are consistent with the possibility that the MAR and/or DNA methylation negatively regulate(s) Vkappa-Jkappa joining during the pre-B cell stage of development.

Symptoms of communication breakdown in dementia: carers' perceptions.

The breakdown of applied communication skills associated with the cognitive-communication deficit of dementia is poorly documented, as is carers' awareness of the nature of the problems. This study considered 32 symptoms which may be associated with breakdown of communication. Perceptions of these symptoms by carers of 79 community-living dementia patients were compared with perceptions of family/close friends of a comparable group of 76 control subjects. Communication skills of dementia patients were perceived to be significantly more impaired than those of control subjects. Areas of greatest discrepancy between performance of dementia and control groups included 'Asking the same question a number of times', 'Difficulty following conversation when a group of people are talking', 'Trouble keeping a conversation going' and 'Struggling to think of the names of people and places'. Eleven of the 32 symptoms were reported as occurring 'fairly often', 'usually' or 'always' in at least 50% of the dementia patients. Identification of specific factors likely to contribute to breakdown of communication in dementia will enable involved professionals to guide carers towards a better understanding of the problems. This should help to alleviate domestic stress thereby enhancing the quality of life of patients and their carers.

Immunoglobulin kappa gene enhancers synergistically activate gene expression but independently determine chromatin structure.

Previous studies have located transcriptional enhancer elements within both the intron and 3'-region of the mouse kappa immunoglobulin gene. Here we address the role of these two enhancers in specifying gene activity and specific chromatin structures. MOPC41 kappa gene constructs, either intact or containing deletions of one or both enhancers, were introduced into S194 mouse plasmacytoma cells for transient and stable expression studies. Transient expression assays revealed that the basal level expression exhibited by enhancerless constructs was activated 100-200-fold by the two enhancers together in a synergistic fashion. A similar trend was observed when both enhancers were present in stably integrated constructs, although the synergy was less pronounced. Analysis of DNase I hypersensitive sites in the chromatin revealed that stably integrated constructs established hypersensitive sites about the enhancer sequences. These sites demonstrated the same nuclease susceptibility as those associated with the endogenous gene(s), and their establishment was independent of the presence of the other enhancer. Thus, although both enhancers are required for maximal gene expression, the elements act independently in determining specific chromatin structures.

Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2.

A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04. The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g. However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein. The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1 alpha subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.

Distribution of catalase and its modulation by 12-O-tetradecanoylphorbol-13-acetate in murine dermis and subpopulations of keratinocytes differing in their stages of differentiation.

Topical treatment of female SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced both dermal and epidermal catalase-specific activities 38% and 51% within 6 h and 18 h of promoter application, respectively. Dermal catalase activity recovered to control levels within 72 h of treatment whereas epidermal catalase activity remained suppressed. Activity measurements were also made in four subpopulations of keratinocytes prepared by Percoll gradient centrifugation that differed in their stages of differentiation. Catalase-specific activity increased with keratinocyte maturity and ranged from 45-54 U/mg protein for basal cell preparations to 252 U/mg protein for granular-squamous cell preparations. Pretreatment of the epidermis for 16-18 h with TPA (2 micrograms) uniformly reduced catalase-specific activity 46-52% in all keratinocyte subpopulations prepared by Percoll gradient centrifugation. Similarly, plots of catalase units per cell versus extracted protein per cell suggested 55-60% decreases in catalase activity in basal and spinous cell keratinocytes of TPA treated epidermis. Furthermore, catalase-specific activity in homogenates of whole epidermis (144-182 units/mg protein) was most similar to the activity of the granular/squamous keratinocyte subpopulation. Collectively, these studies suggest that: (i) TPA reduces the capacity for H2O2 detoxification by catalase throughout the epidermis; and (ii) activity measurements on unfractionated epidermal preparations may not be representative of the basal cell keratinocyte population.