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H Cooke

Publications and source records attributed to H Cooke.

At least 19 recordsLinked to original sources
PubMed1998

Assay of centromere function using a human artificial chromosome.

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Autoantigens↗
PubMed1998

Construction of YAC-based mammalian artificial chromosomes.

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

Animals↗
PubMed1998

Engineering mammalian chromosomes.

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.

Animals↗
PubMed1997

Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm.

The association of abnormal spermatogenesis in men with Y chromosome deletions suggests that genes important for spermatogenesis have been removed from these individuals. Recently, genes encoding two putative RNA-binding proteins (RBM and DAZ/SPGY) have been mapped to two different regions of the human Y chromosome. Both of these genes encode proteins that contain a single RNA recognition motif and a (different) internally repeating sequence. Y-linked RBM homologues are found in all mammalian species. We have raised an antiserum to RBM and used it to show that RBM is a nuclear protein expressed in fetal, prepubertal, and adult male germ cells. The distribution of RBM protein in the adult correlates with the pattern of transcriptional activity in spermatogenesis, suggesting that RBM is involved in the nuclear metabolism of newly synthesized RNA. RBM sequences are found on both arms of the Y chromosome making genotype-phenotype correlations difficult for this gene family. To address the location of the functional genes and the consequences of their deletion, we examined a panel of men with Y chromosome deletions and known testicular pathologies using this antiserum. This approach enabled us to map a region of the Y chromosome essential for RBM expression. In the absence of detectable RBM expression we see stages of germ cell development up to early meiosis, but not past this point into the haploid phase of spermatogenesis.

Adult↗
PubMed1996

An RBM homologue maps to the mouse Y chromosome and is expressed in germ cells.

We have isolated a murine homologue of the human Y-linked RBM genes (previously termed YRRM), a gene family implicated in spermatogenesis and which encodes proteins containing an RNA recognition motif. A number of very similar copies of this gene (called Rbm) are present in the mouse. These mouse homologues are also Y-encoded, mapping on the short arm of the chromosome, proximal to Sry. Expression is confined to the testis, specifically the germ line on the basis of lack of expression in the germ-line negative testes of adult sex-reversed mice. The timing of Rbm transcription is regulated, with fetal message levels reaching a peak at 15 d.p.c. Transcripts are clearly detectable by 4 days after birth and reach their highest level at 14 d.p.p. which is the time at which the Y chromosome condenses during meiotic prophase. These results suggest that Rbm is functionally involved in germline RNA metabolism.

Amino Acid Sequence↗
PubMed1996

Y chromosome microdeletions and male subfertility.

Men with azoospermia and severe oligozoospermia have been investigated by molecular probing of the long arm of the Y chromosome. We find microdeletions affecting various parts of the long arm of the Y chromosome in approximately 10% of men with non-obstructive azoospermia and severe oligozoospermia but not in a fertile comparison population. This work needs further confirmation in different countries and different racial groups but it would appear that microdeletions (and presumably genetic defects) are commonly associated with defects of spermatogenesis. These findings have implications for the management of severe male subfertility with in vitro fertilization/intracytoplasmic sperm injection.

Case-Control Studies↗
PubMed1994

Sequence homologies and linkage group conservation of the human and mouse Cenpc genes.

Using a previously identified human CENPC cDNA fragment, we have isolated cDNA clones corresponding to the complete mouse Cenpc coding sequence. Using these cDNAs as probes to genomic libraries, we have isolated genomic clones corresponding to the mouse and human genes and also to a mouse pseudogene. In situ hybridization mapping of these genes reveals that the human gene maps to 4q12-q13.3 and the mouse gene to 5E2-E5. These sites are in a region of linkage group conservation between the two species. Secondary sites are present in man on chromosome 12q21.2-q21.33 and in mouse on chromosome 2B. This mouse secondary site is a pseudogene on the basis of DNA sequence. These secondary sites are not syntenic in the two species.

Amino Acid Sequence↗
PubMed1994

Students' evaluation of the process of conducting a patient assessment.

A Qualitative Evaluation Strategy was adopted to explore an early curriculum experience of undergraduate pre-registration student nurses. Open-ended narratives, in the style of Illuminative Evaluation (Parlett & Hamilton 1972), were used to encourage students to reflect upon their experience of conducting their first patient assessment using a model of nursing. A content analysis of the students' reflections is presented and discussed with reference to studies whose findings they corroborate and support. Aspects of the learning experience which influence the development of nursing skills were elucidated, together with the learning processes involved in the acquisition of patient assessment skills. Evaluating the context of the students' learning environment revealed some of the realities of learning nursing 'in the field', a lack of role models in clinical areas and the persistence of a theory-practice gap. In addition, this study shed light on the process of professional socialisation in student nurses. Encouraging reflection helped the students recognise how much they had learnt, and enabled us to evaluate our effectiveness as learning facilitators. An electic approach combining both qualitative and quantitative paradigms is advocated for holistic evaluation of the curriculum. Inferences from this evaluation may have particular relevance for designers and teachers of Project 2000 style courses.

Education, Nursing, Baccalaureate↗
PubMed1993

Why teach sociology?

This paper examines the arguments in favour of the inclusion of sociology in the nursing curriculum. It contains an analysis of the literature on sociology teaching in nursing education and briefly compares this with sociology teaching in the medical curriculum. The discussion draws attention to the ways in which the professional concerns and ambitions of nursing constrain the content and organisation of the sociology curriculum. Finally it argues for a more critical and theoretically informed sociology for nurses.

Curriculum↗
PubMed1993

Boundary work in the nursing curriculum: the case of sociology.

A discussion of the boundaries between nursing and sociology is contained in this paper. The creation of nursing as an academic subject is discussed and compared with the creation of academic geography. The creation of academic subject involves 'boundary work' in which power and legitimacy are conferred on some forms of knowledge and not others. Boundary work enables a discipline to stake out a claim to its legitimate territory and the resources that go with it. In a practice discipline such as nursing, the boundaries between nursing and supporting subjects, such as sociology and physiology, create problems of transfer of learning. This has implications for curriculum design. Bernstein's work on educational transmissions offers useful insights. He suggests a distinction between educational knowledge codes. 'Collection' codes involve strong boundaries between subjects, 'integrated' codes imply weak boundaries. The implications of the move to an integrated code in nursing are discussed. The existence of an integrated code implies a 'strong ideological consensus' within a discipline. In nursing this entails a belief in the 'individualized care' of the patients. This is incompatible with the sociological understanding of nursing.

Curriculum↗
PubMed1992

hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn.

We have previously isolated a protein from mouse liver nuclei that specifically binds to single stranded (TTAGGG)n repeats. TTAGGG is the telomeric repeats of mammals and we therefore named the new protein single stranded telomere binding protein (sTBP). Further studies now identify sTBP as heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 on the basis of amino acid sequence determination and antibody reactivity. A2 and B1 form a major part of the protein component of hnRNP particles and are abundant nuclear proteins. Unexpectedly, A2/B1 has a high specificity for binding to the RNA equivalent of TTAGGG, UUAGGG, but under the same conditions does not appear to have a strong affinity for a number of other RNA species.

Amino Acid Sequence↗
PubMed1992

Lambda CM8, a human sequence with putative centromeric function, does not map to the centromere but is present in one to two copies at 9qter.

A DNA fragment isolated from a human genomic library, was reported to be present at all human centromeres and present at 16-32 copies per genome. Reintroduction of this DNA into mammalian cells as a concatenated phage clone gave rise to dicentric chromosomes which gave rise to a new, stable, chromosome. Taken together these observations could mean that this DNA is part of a native centromere. We have reexamined the location and copy number of this sequence and find it to be present at 1-2 copies per genome with a single site of in situ hybridisation at 9qter.

Blotting, Southern↗