56 research outputs found
Recombining overlapping BACs into a single larger BAC
BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. RESULTS: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. CONCLUSION: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones
Recommended from our members
[Research Summary] Fit for Work: final report of a process evaluation
This report presents the fndings from the process evaluation of Fit for Work. Fit for Work was an occupational health assessment and advice service looking to address long-term sickness absence. The assessment service was for employees who were on (or at risk of entering) long-term sickness absence, defned as four weeks or more, via a referral through their General Practitioner (GP) or their employer. Participation was entirely voluntary. Employees giving their consent took part in a biopsychosocial assessment, which were primarily conducted by telephone. After assessment, a Return to Work Plan (RtWP) would be produced, with recommendations for self-care, workplace adjustments, and/or signposting to further specialist support and therapy services to assist the employee’s return to work. With the employee’s consent, the RtWP could be shared with their employer and/or GP. The service was funded by the Government and was delivered in England and Wales by Health Management Limited (HML) and in Scotland via an agency agreement with the Scottish Government.
The process evaluation aimed to determine whether the Fit for Work service had been implemented as designed, and whether the design met the policy intent to provide support for those on long-term sickness absence to stay in employment.
The research programme was conducted between September 2015 and May 2017 and consisted of: an analysis of management information; 72 in-depth qualitative interviews with employees, employers and GPs; a telephone survey of 504 employers that had had contact with the service; a telephone survey of 1,045 employees that had been discharged from the service and a follow-up survey of 492 of them (only those who received an assessment and who gave permission to be re-contacted for further research). Management information was analysed for employees referred to and discharged from the Fit for Work service between October 2015 and December 2016 inclusive. As this analysis did not cover the whole period of the service, fndings could differ from the overall management information. In some instances, due to small sample sizes, apparent differences between groups (e.g. between countries) may not be statistically signifcant and therefore should be viewed with caution. Following very low referrals, it was announced that the Fit for Work assessment service would come to an end in England and Wales on 31 March 2018 and 31 May 2018 in Scotland. However, employers, employees and GPs will continue to have access to the same Fit for Work helpline, website and web chat, which offer general health and work advice as well as support on sickness absence.
This process evaluation was undertaken before the decision was taken to close the service
Choice of syntactic structure during language production: The production of unbounded dependencies
During language production, conceptual messages are encoded into a target
language and articulated. Existing models of language production assume several
stages of processing including a conceptual level, a level where lexical selection and
syntactic processing occurs and a level where morphological and phonological
features are added ready for production (e.g. Levelt et al., 1999). Previous research
has considered how lexical and syntactic information could be stored via lemma
(Kempen & Huijbers, 1983), syntactic nodes (Levelt at el., 1999) and combinatorial
nodes (Pickering & Braingan, 1998), but little is understood about how syntactic
structures are selected. This thesis examines how constituent structures are selected
by investigating choice of structure in unbounded dependencies such as Which jug
with the red spots is the nun giving the monk? and how this is affected by factors such
as verb-subcategorisation preferences and global sentence structure complexity.
A series of language production experiments investigate how global
structure complexity and verb-subcategoricatisaion preferences affect choice of
syntactic structure at the clause level in unbounded dependencies. A picture
description task reveals an unusual preference for the dispreferred passive voice
structure as a result of global structural complexity. Sentence recall experiments
demonstrate that both global structural complexity and verb-subcategorisation
preferences can affect choice of structure and that competition between these factors
decides the final structure. Finally, syntactic priming experiments show that
processing mechanisms are shared between simple matrix clause structures and
unbounded dependency clause structures, but that the influence of these shared
mechanisms vary between the different structure types. This could be attributed to a
modal of processing where choice of structure is decided by competition between
structure representations which are influenced by different factors in different
global syntactic conditions.
The results suggest that choice of syntactic structure is decided through
competition between possible structures. These possible structures may receive further activation or inhibition from other factors such as global structural
complexity or verb-subcategorisation preferences and thematic fit. Global structural
complexity may influence structure preferences through increased processing load
or through attempts to integrate the clause structure with another global structure.
Thematic role arguments may influence structure through a preference that
syntactic roles fit with specified thematic roles. (e.g. experiencer as subject). This
model assumes parallel processing of possible structures and individual structures
within a complex larger structure. It also assumes an incremental model of
processing which attempts to integrate structures as soon as possible
Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines.
We demonstrate that transfer of a yeast artificial chromosome (YAC) containing 230 kb of the human beta-globin locus into mouse erythroleukemia cells by fusion results in correct developmental regulation of the human beta-like globin genes. Additionally, we show that early after hybrid formation, human embryonic epsilon- and fetal gamma-globin genes are coexpressed with the adult beta gene but that after 10-20 weeks in culture, globin gene expression switches to predominantly adult. Thus, in contrast to shorter gene constructs, the globin genes of the beta-globin locus YAC are regulated like the chromosomal globin genes. These results indicate that transfer of YACs into established cell lines can be used for the analysis of the developmental control of multigenic and developmentally regulated human loci
Mapping investment in adult skills – which individuals, in what learning and with what returns?
The report explores how public investment in adult skills through the Adult Skills Budget compares with other funding streams, such as investment by employers. This review took place before the apprenticeships levy on large employers
Connecting Communities:Process Evaluation: Final Report
Connecting Communities was a voluntary employment support programme funded by the Department for Work and Pensions (DWP) and procured and overseen by the West Midlands Combined Authority (WMCA). It was tested across nine geographically defined neighbourhoods (also called ‘lots’) and ran for three and a half years. This period included the Covid-19 pandemic, which impacted on programme delivery, employment opportunities and everyday life more generally. The nine lots were located across the WMCA area: Birchills Leamore; Batchley and Brockhill; Cannock North; Washwood Heath; Shard End; Chemsley Wood; Binley and Willenhall; Camp Hill; and Glascote.Emphasising intensive, personalised, and context-specific support, the programme sought to: (a) build social networks to foster positive behavioural and attitudinal changes towards work; (b) increase employment; and (c) work with local businesses to bolster the recruitment and progression of disadvantaged individuals. This evaluation identifies the factors that influenced employment and progression outcomes for participants, as well as the strengths and weaknesses of the place-based approach to employment support more broadly. The study drew on qualitative data from observational site visits, in-depth interviews, and analysis of management information and claims data. An impact evaluation and assessment of cost-effectiveness will be published separately.<br/
Connecting Communities:Process Evaluation: Final Report
Connecting Communities was a voluntary employment support programme funded by the Department for Work and Pensions (DWP) and procured and overseen by the West Midlands Combined Authority (WMCA). It was tested across nine geographically defined neighbourhoods (also called ‘lots’) and ran for three and a half years. This period included the Covid-19 pandemic, which impacted on programme delivery, employment opportunities and everyday life more generally. The nine lots were located across the WMCA area: Birchills Leamore; Batchley and Brockhill; Cannock North; Washwood Heath; Shard End; Chemsley Wood; Binley and Willenhall; Camp Hill; and Glascote.Emphasising intensive, personalised, and context-specific support, the programme sought to: (a) build social networks to foster positive behavioural and attitudinal changes towards work; (b) increase employment; and (c) work with local businesses to bolster the recruitment and progression of disadvantaged individuals. This evaluation identifies the factors that influenced employment and progression outcomes for participants, as well as the strengths and weaknesses of the place-based approach to employment support more broadly. The study drew on qualitative data from observational site visits, in-depth interviews, and analysis of management information and claims data. An impact evaluation and assessment of cost-effectiveness will be published separately.<br/
- …