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R Lindeman

Publications and source records attributed to R Lindeman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.

We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).

Bacteriophage lambda↗
PubMed1993

Molecular characterisation of Vietnamese HPFH.

A novel 30 kb deletion of the beta-globin gene cluster associated with the phenotype of hereditary persistence of fetal hemoglobin (HPFH) is described in two unrelated individuals of Vietnamese background. The Vietnamese G gamma A gamma HPFH deletion has a unique 5' breakpoint 3.5 kb downstream of the delta-globin gene. The 3' breakpoint lies approximately 8 kb upstream from the HPFH-3 breakpoint (Henthorn et al., 1986) and in the region of the 3' breakpoints of HPFH-4 (Saglio et al., 1986), German and Belgian G gamma+ (A gamma delta beta)zero-thalassemias (Anagnou et al., 1988; Losekoot et al., 1991). Characterisation of the 3' breakpoint in the present study has enabled more precise localisation of other deletion breakpoints at this locus. Further evidence is provided that the 3' breakpoint region contains functionally important sequences and that the juxtaposition of these sequences to the gamma-globin genes is a significant factor in the increased fetal hemoglobin levels.

Adult↗
PubMed1993

Comparative studies of nondeletional HPFH gamma-globin gene promoters.

The -198 T-->C and the -175 T-->C transitions involving the proximal gamma-globin gene promoter are associated with the hereditary persistence of fetal hemoglobin (HPFH) phenotype and have been demonstrated to increase promoter activity in erythroid cells using transient and stable transfection systems. The above base changes are thought to alter the binding of different transcription regulatory proteins. Another mutation of the proximal gamma-globin promoter, -158 C-->T, has been less clearly linked to the HPFH phenotype but has been associated with increased G gamma activity. In the present paper, the -198 T-->C, -175 T-->C and -158 C-->T mutations both singly and in various combinations were evaluated by an in vitro expression assay. gamma-Globin promoters were transfected by electroporation into K562 human erythroleukemia cells and their activity measured in a human growth hormone (hGH) reporter gene assay. A novel cotransfectant was used to assess transfection efficiency. Results confirmed the previously reported upregulation of gamma-globin activity with the -198 T-->C and -175 T-->C HPFH mutations and a cooperative effect on promoter activity when both these mutations are present in cis. No effect of the -158 C-->T mutation was seen either alone or in combination with the -175 T-->C and -198 T-->C mutations.

Base Sequence↗
PubMed1992

Australian beta zero-thalassaemia: a high haemoglobin A2 beta zero-thalassaemia due to a 12 kb deletion commencing 5' to the beta-globin gene.

A large novel deletional beta zero-thalassaemia mutation associated with unusually high levels of haemoglobin A2 in heterozygotes is described in an Australian family. The deletion was characterized by restriction enzyme analysis followed by PCR amplification and sequencing of the breakpoint region. Australian beta zero-thalassaemia extends from 835 basepairs (bp) 5' to the cap site of the beta-globin gene downstream for 12.023 kb. This deletion, similar to previously described deletional beta zero-thalassaemias associated with high Hb A2, removes sequences 5' to the beta-globin gene promoter and emphasizes the functional importance of the 5' beta-globin region in eliciting the unusually high Hb A2 phenotype.

Base Sequence↗
PubMed1992

An A gamma globin promoter (four base-pair deletion) mutant shows linked polymorphic changes throughout the A gamma gene.

The A gamma fetal globin genes from a large Australian kindred with nondeletional A gamma hereditary persistence of fetal hemoglobin (HPFH) were cloned and sequenced. The -198 T----C mutation (British type HPFH) was demonstrated upstream of the A gamma gene on one allele. On the other allele, a 4-deletion was identified -222 to -225 bp upstream from the cap site. The 4-bp deletion allele was associated with a number of variations in the A gamma gene sequence: anA gamma T transition, in the second exon (T----C at +402 relative to the cap site); a HindIII polymorphism in the second intron; and a G gamma-like sequence in the 3' untranslated region (TCAC in place of CTCT, creating a SacI site). An association between the -222 to -225 deletion, the A gamma T polymorphism, and the second intron HindIII polymorphism has previously been reported. In addition, linkage of the HindIII polymorphism with the G gamma-like sequence in the 3' untranslated region of A gamma has also been described. The case described here is unique, with all four changes present in the one A gamma gene. It is also noteworthy because there is simultaneous occurrence of high (HPFH) and low (-222 to -225 deletion) expression mutants in the same patient. Despite the presence of the 4-bp deletion, the resulting hematological phenotype remained that of HPFH. When the 4-bp deletion promoter was studied in a K562-cell transient expression assay, there was found to be no statistically significant reduction in activity compared to the control A gamma promoter. The possible reasons for the observed differences between the in vivo and in vitro activity of this mutation are discussed.

Alleles↗
PubMed1991

A de novo unbalanced reciprocal translocation identified as paternal in origin in the Prader-Willi syndrome.

Interstitial cytogenetic deletions involving the paternally derived chromosome 15q11-13 have been described in patients with the Prader-Willi syndrome (PWS). We report a child with PWS and a de novo unbalanced karyotype -45,XY,-9,-15,+der(9)t(9;15)(q34;q13). Molecular studies with the DNA probe pML34 confirmed that only a single Prader Willi critical region (PWCR:15q11.2-q12) copy was present. Hybridisation of patient and parental DNA with the multi-allelic probe CMW1, which maps to pter-15q13, showed that the chromosome involved in the translocation was paternal in origin. This is the first example of a paternally-derived PWCR allele loss caused by an unbalanced translocation that has arisen de novo.

Blotting, Southern↗
PubMed1991

Polymerase chain reaction (PCR) mutagenesis enabling rapid non-radioactive detection of common beta-thalassaemia mutations in Mediterraneans.

The IVS-1-110 (G----A) and IVS-1-1 (G----A) mutations occur in approximately 33% and 9% respectively of beta-thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)-amplified material by allele-specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3' base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS-1-110) and HinfI (IVS-1-1) produced different fragments on electrophoresis. The above strategy was validated by allele-specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS-1-110, codon 39 and IVS-1-1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.

Base Sequence↗
PubMed1991

Molecular and cytogenetic studies of the Prader-Willi syndrome.

Twenty-seven subjects with the Prader-Willi syndrome (PWS) were studied. Sixteen (59%) had a cytogenetic deletion involving chromosome 15q11-13. Nine were non-deletional and two patients had structural rearrangements of chromosome 15: 47,XY, + del(15)(pter----q12), var(15)(p11) and 45,XX,t(14q15q). At the DNA level, a greater proportion of patients (74%) showed loss of one chromosome 15q11-13 allele using a combination of densitometry and RFLP analysis. Deletion sizes were variable with 13 of 20 detectable both cytogenetically and with probe pML34 (D15S9). The remaining seven had microdeletions at the pML34 locus. Heterogeneity was further seen in three subjects who had cytogenetic deletions but normal DNA studies. In one patient there was evidence of a duplication at the pML34 locus. A new molecular rearrangement was identified with probe p3.21 (D15S10) in two patients and their mothers. Fifteen family studies were performed. In all 10 families where there was a molecular deletion, this was shown to have arisen de novo. DNA mapping confirmed that the paternal 15q allele was lost in three patients with PWS.

Adolescent↗
PubMed1991

Utility of the polymerase chain reaction (PCR) for prenatal diagnosis of genetic disease.

Gene amplification by the polymerase chain reaction (PCR) has been applied to prenatal diagnosis for alpha and beta thalassemias (1 and 5 cases respectively), Hemoglobin (Hb) Lepore/beta thalassemia (1 case) and cystic fibrosis (14 cases). Chorionic villus samples were obtained in the tenth week of pregnancy and DNA analysed in parallel with conventional gene mapping. Direct diagnosis of the common Mediterranean beta-thalassemia mutations (IVS-1-110 and codon 39), Hb Lepore, and the delta F508 mutation causing cystic fibrosis was achieved by hybridization of amplified material with pairs of allele-specific oligonucleotide (ASO) probes or by restriction enzyme digestion of PCR products. Results were confirmed by DNA mapping. Definitive diagnosis or exclusion of an affected fetus was possible in 17 of 21 cases thus examined. PCR reduces the time required for prenatal diagnosis. DNA contamination is a potential source of error.

Alleles↗
PubMed1990

Exclusion of beta-thalassaemia by biopsy and DNA amplification in mouse pre-embryos.

Exclusion of beta-thalassaemia in mice was undertaken by pre-implantation DNA diagnosis. Biopsies of approximately five cells were obtained from mouse pre-embryos. DNA prepared from this material was amplified by the polymerase chain reaction (PCR), enabling identification of the normal beta-major globin gene in 11 out of 20 cases where it was known to be present. The expected negative result was obtained in 14 out of 16 homozygous thalassaemic embryos, with two false positives. Results are promptly available following PCR, thereby facilitating re-implantation of embryos which have had homozygous beta-thalassaemia excluded. The mouse model illustrates a useful approach to pre-implantation diagnosis of genetic disease in the human.

Animals↗
PubMed1990

Experience of a molecular genetics service in prenatal diagnosis by DNA analysis.

Prenatal diagnoses of the genetic disorders alpha, beta thalassemia, HbS, Hb Lepore, hemophilia and cystic fibrosis were sought in 88 cases. Six unsuccessful attempts at diagnosis resulted from DNA polymorphisms which were only 50% informative (four cases) and prenatal diagnoses which had been undertaken before it was known whether DNA polymorphisms in family studies were informative (two cases). The most frequent indications for prenatal diagnosis were the hemoglobinopathies although requests for exclusion of cystic fibrosis formed the majority during 1989. Strong linkage disequilibrium between the cystic fibrosis defect and its associated DNA polymorphisms facilitated detection of this disorder. Late presentations among patients with beta thalassemia and hemophilia and the necessity for more specialised genetic counselling were the commonest problems encountered.

Chromosome Mapping↗
PubMed1987

Demonstration of lymphoid antigens in decalcified bone marrow trephines.

A panel of antibodies recognising lymphoid and epithelial antigens in formalin fixed, paraffin embedded sections was applied to a series of 54 bone marrow trephines decalcified by formic or edetic acids. Normal trephines and cases infiltrated by myeloid, lymphoid, and epithelial tumours were included. Patterns of reactivity were distinct and allowed the different diseases to be distinguished. All lymphoid tumours expressed leucocyte common antigen, with B cell tumours staining with MB1 and MB2, and T cell tumours staining with MT1 and UCHL1. T cell acute lymphoblastic leukaemia (ALL)/lymphoblastic lymphoma all stained with MT1, but some were negative with UCHL1. B cell ALL/lymphoblastic lymphoma also stained with MT1, but could be distinguished by its reactivity with MB1 and MB2. Reed-Sternberg cells did not stain with any reagent. Normal and neoplastic myeloid cells stained with MT1. Carcinomas stained with CAM 5.2 but were negative for lymphoid markers except MB2 staining in some cases. A case of neuroblastoma could be distinguished from ALL/lymphoblastic lymphoma by its lack of reactivity with all antileucocyte antibodies and its staining with antineurone specific enolase. Although not ideal, if used together, this panel of reagents may usefully be applied to routinely fixed and processed, decalcified bone marrow trephines.

Antibodies, Monoclonal↗
PubMed1979

Development of the segmental innervation of the chick forelimb.

A number of recent studies have shown that during embryonic development the initial innervation of a target structure may be made up, in part, by axons which do not form part of the mature innervation of that structure. In the present study we have examined the motor innervation of the major muscles of the chick forelimb at different stages of development using HRP-uptake-labelling of motoneurons, electrophysiological recording and measurement of muscle contraction. In the mature White Leghorn chick the major contribution to the motor innervation of the forelimb is from spinal segments 14, 15 and 16. Using the HRP-labelling technique we have shown that at stages 26-29 of development motoneurons in segments 12-17 have axon terminals in the presumptive biceps muscle. Between stages 30 and 35, however, the axon terminals arising from segments 12, 13, 16 and 17 are lost, leaving the mature innervation from segments 14 and 15. We have also observed the loss of innervation of the biceps muscle by segment 16 using electrophysiological recording of compound action potentials in the biceps nerve and by measurement of the local contraction of the biceps muscle in response to stimulation of the segmental nerves. Similar changes in the innervation of the triceps, extensor metacarpi radialis, flexor carpi ulnaris and flexor digitorum profundus muscles have also been observed. These results are discussed in relation to the hypothesis that (i) the motoneuron pools and muscles in the developing spinal cord and forelimb are matched, (ii) that some axons which arrive in a particular muscle during early development are unable to form a stable connexion and (iii) that the inability of an axon terminal to form a stable connexion in a muscle results in the death of the motoneuron. Intracellular recording from muscle cells at stage 35 shows that the synaptic site on each cell is innervated by about three separate axons. Over the next few stages, however, all but one of the innervating axons is lost. From our contraction studies it is clear that the removal of the excess axon terminals after stage 35 is not associated with the establishment of the mature segmental innervation pattern of the muscle.

Action Potentials↗
PubMed1979

Segmental innervation of the chick forelimb following embryonic manipulation.

A number of studies have shown that the segmental innervation of some muscles in the developing limb undergoes some modification during the earliest stages of ontogeny. These observations can be interpreted in support of the hypothesis that the motor axons and muscles are matched during this period of development. As a further test of this suggestion we have made a quantitative examination of the motor innervation of the chick forelimb under conditions of controlled abnormal development. Embryos were surgically manipulated at stages before the motor axons invade the limb. The operations were controlled such that forelimbs were induced with segments deleted or reduplicated or simply that a segment of the spinal cord had been deleted. In preparations with abnormal limbs the motor innervation of the muscles present was the same as for those muscles in the normal limb. Where a spinal segment had been deleted the limbs developed normally and their innervation was completed by the remaining brachial segments. These results suggest that any particular matching property of a developing muscle does not develop as a consequence of its position in the limb relative to those segments of the limb proximal to it. Furthermore, that some muscles which are normally innervated by two spinal segments can be completely innervated by one of those spinal segments, in the absence of the other, suggests that any matching between growing axons and developing muscle cells is hierarchical rather than strictly all-or-nothing.

Animals↗