R. G. H. Cotton

Current methods of mutation detection

Mutation detection is important in all areas of biology. Detection of unknown mutations can involve sequencing of kilobases of DNA, often in many patients. This has lead to the development of methods to screen DNA for mutations as well as methods to detect previously described mutations. This review discusses current methods used for such purposes with special emphasis on genetic diseases of humans. However, savings can be made by similar means in other areas of biology where repetitive or extensive sequencing for comparative purposes needs to be done. This review covers the methods used for detection of unknown mutations, namely the ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single-strand conformation polymorphism, heteroduplex and sequencing methods. Once mutations have been defined they can be searched for repeatedly by methods referred to as diagnostic methods. Such methods include allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites. We can now choose from a range of excellent methods, but the choice will usually depend on the background of the laboratory and/or the application in hand. Screening methods are evolving to more satisfactory forms, and the diagnostic methods can be automated to screen whole populations inexpensively.

Loss of brainstem serotonin- and substance P-containing neurons in Parkinson's disease.

Using postmortem immunohistochemical analysis, we have identified degeneration of several different neuronal cell groups in the brainstem of patients dying with idiopathic Parkinsons disease. We report the first chemically identified loss of presumed serotonin neurons in the median raphe nucleus of the pons and of substance P-containing preganglionic neurons in the dorsal motor vagal nucleus. This evidence is concordant with other evidence that the primary neuropathological process is not confined either to a single pathway or to neurons containing a particular transmitter. Rather it appears that Parkinsons disease affects several classes of neurons in localized areas of the brainstem.

Distribution, morphology and number of monoamine-synthesizing and substance P-containing neurons in the human dorsal raphe nucleus

The distribution, morphology and number of serotonin-, catecholamine- and substance P-containing neurons in the human dorsal raphe nucleus were studied. Parallel series of sections were prepared from 10 human brainstems obtained at autopsy from patients without neurological disease aged between 42 and 88 years. The neurons were identified using immunohistochemistry with antibodies raised against phenylalanine hydroxylase (tryptophan hydroxylase-containing, serotonin neurons), tyrosine hydroxylase (catecholamine neurons) and substance P. A reference series of Nissl-stained sections was also prepared and data published separately were used to delineate the subnuclear divisions of the dorsal raphe nucleus and to establish the total number of neurons in each subnucleus. The following principal findings emerged. (1) Serotonin-synthesizing neurons are present in all regions of the dorsal raphe nucleus and their total number is 165,000 +/- 34,000. The same types of neurons as those seen in Nissl material characterize each of the five subnuclei (caudal, dorsal, ventral, ventrolateral and interfascicular). (2) Substance P-containing neurons mostly occupy the rostral part of the nucleus and their number is 74,600 +/- 17,600. (3) Catecholamine cells are only found in the rostral part of the dorsal raphe nucleus and their number is 5600 +/- 3400. (4) In the ventral and interfascicular subnuclei the combined number of serotonin-synthesizing and substance P-containing neurons exceeds the total number of Nissl-stained neurons suggesting that serotonin and substance P co-exist in a substantial part of the cell population of the dorsal raphe nucleus. This is further supported by the highly similar morphology and size of these neurons. It is concluded that there are demonstrable chemical differences between the various subregions of the human dorsal raphe nucleus. These differences are in harmony with the results of hodological studies in animals, which have demonstrated differential projection pathways emerging from this nucleus.

Brain stem serotonin-synthesizing neurons in Alzheimer's disease: a clinicopathological correlation

SummaryThe location and number of brain stem serotonin-synthesizing neurons were analyzed in 11 patients with Alzheimers disease (AD) and 5 agematched controls using immunohistochemical techniques. In addition, the number of neuritic plaques and neurofibrillary tangles in the cortex and brain stem raphe was evaluated, as was the number of Nissl-stained raphe neurons. AD patients could be classified into two groups based on their raphe pathology; patients with such pathology (AD+) and those without (AD−). The number of large raphe neurons correlated significantly with the number of serotonin-synthesizing neurons in control material, indicating that all large neurons were serotonergic. This relationship was not apparent in AD+ patients, in whom the number of serotonin-synthesizing neurons correlated with the number of neurofibrillary tangles in the raphe of these patients. This indicates that in AD+ patients the serotonin-synthesizing neurons were selectively affected. There was no correlation between raphe and cortical pathology or raphe pathology and patient sex, age, mini-mental score or depression score, even when such scores were weighted for the interval between testing and death. There was a trend for the raphe pathology to correlate with the age of onset and duration of dementia and the Blessed dementia score in AD+ patients. Most AD+ patients with severe raphe lesions had clinical dementia only, while AD− patients had additional clinical features. The raphe lesions were more dramatic in AD+ patients with a rapid progression of symptoms.

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.

Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A

Abstract: The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.

The distribution of neuropeptide Y-like immunoreactive neurons in the human medulla oblongata

We have described the distribution of neuropeptide Y-like immunoreactive neurons in the medulla oblongata of the adult human. The majority of neuropeptide Y-like immunoreactive cells were found in four regions of the medulla: the ventrolateral reticular formation, the dorsomedial medulla, the secondary sensory nuclei and the rostral raphe nuclei. The morphology of neuropeptide Y-like immunoreactive cells varied in each of these regions. In the ventrolateral reticular formation, the labelled neurons were round and pigmented caudal to the obex but elongated and non-pigmented rostral to the obex; in the dorsomedial medulla, they were triangular and pigmented caudal to but not rostral to the obex; in the secondary sensory nuclei, they were multipolar, non-pigmented and significantly smaller than in the other areas; in the rostral raphe nuclei, they were bipolar and non-pigmented. Colocalization studies revealed that many neuropeptide Y-like immunoreactive cells also synthesize monoamines, consistent with conclusions based on a quantitative comparison of their distributions. Neuropeptide Y-like immunoreactivity was present in about 25% of presumed noradrenaline-synthesizing cells in the caudal ventrolateral medulla (corresponding to the A1 region); about 50% of adrenaline- and 70% of presumed serotonin-synthesizing cells in the rostral ventrolateral medulla (C1 and B2-3 regions); 90-100% of presumed noradrenaline-synthesizing cells in the dorsomedial medulla at and above the obex (A2 region); about 50% of adrenaline-synthesizing cells in the rostral dorsomedial medulla (C2 region); about 5% of presumed serotonin-synthesizing cells in the rostral raphe nuclei (B2-3 region). The largest of these groups was the presumed serotonin-synthesizing cells that contained neuropeptide Y-like immunoreactivity in the rostral ventrolateral medulla. This is the first report of such a cell group in the medulla of any mammal, and emphasizes the neuroanatomical differences between humans and other species.

Biochemical Defect of the hph‐1 Mouse Mutant Is a Deficiency in GTP‐Cyclohydrolase Activity

A hyperphenylalaninemic mouse mutant, hph‐1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid‐dihydropteridine reductase activity is also normal. GTP‐cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP‐cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4‐dihydroxyphenylalanine (DOPA) and serotonin.

Tryptophan Hydroxylase Is Phosphorylated by Protein Kinase A

Abstract: The effect of protein kinase A on the catalytic activity and phosphorylation of brain tryptophan hydroxylase was examined. Stimulation of endogenous protein kinase A by cyclic AMP or its analogues, dibutyryl‐cyclic AMP and 8‐thiomethyl‐cyclic AMP, failed to activate tryptophan hydroxylase. The activation of tryptophan hydroxylase by calcium/calmodulin‐phosphorylating conditions was not modified by cyclic AMP. Endogenous protein kinase A phosphorylated a large number of proteins and tryptophan hydroxylase could be identified as one substrate by sucrose gradient centrifugation, immunoprecipitation, and immunoblotting. These results indicate that tryptophan hydroxylase is phosphorylated by protein kinase A in brain and question whether this protein kinase exerts direct regulatory influence over tryptophan hydroxylase activity via phosphorylation.

The use of chemical reagents in the detection of DNA mutations

As the analysis of the human genome proceeds at an ever-increasing pace, many genes have been identified which are the site for mutations responsible for inherited diseases. The identification of the mutations within these genes has become a major application of molecular biology technologies, and to this end a number of mutation detection systems have been developed for use in diagnostic and research laboratories. The uses of these mutation detection systems are in the diagnosis of inherited disease (both prenatal and neonatal) and in an understanding of the function of the affected protein by cataloguing the range of mutations. Two of these mutation detection systems are reviewed here. Both rely on chemical modification of mismatched nucleotides, by either carbodiimide or hydroxylamine and osmium tetroxide. The methods are termed the carbodiimide (CDI) and the Chemical Cleavage of Mismatch (CCM) methods. The history and evolution of the methods is tracked, illustrating the way in which they developed, both as suitable technology became available (for example, the polymerase chain reaction) and as a result of a specific need. The current methodologies are briefly discussed, followed by a discussion of their applications, especially in the realm of disease mutation detection.