A digital thermocouple thermometer

Measurements of the reproducibility of a random selection of copper/constantan thermocouples were made and it was found that they agreed within 1 ° C. Based on this finding, a digital thermocouple thermometer was designed and constructed incorporating a thermocouple linearizer and cold junction compensation. The instrument

Accuracy of the Completed Digital Thermometer

     

2.

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells     

P. I. Liu  K. C. Poon  L. Crook  S. S. Liu  C. C. Hong 《Cryobiology》1979,16(6):513-516

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

Temperature	Indicated	Error
(°C)	temperature	(°C)
	(°C)
?1.95.75	?195	0.75
?77.02	?78	0.38
0	0	0
52.49	53	0.51
Mean		0.413

	Mean (%)
Type of colonies	t Score	P Value	Unfrozen	Frozen
			Erythrocytic	26.283	14.100	2.09	0.059
Granulocytic	23.741	32.917	1.45	0.173
Mixed	49.321	52.700	0.55	0.59

a

$\text{N = 92}$. the presence of pluripotent hemopoietic precursor cells in cryopreserved 0-, 3-, 6-, 9-, and 12-hr postmortem murine bone marrow cells. Apparently, the erythropoietic precursor cells are more sensitive to freezing injury as compared to granulopoietic precursor cells.

     

Hemopoietic activities of cryopreserved murine fresh and postmortem bone marrow cells

The percentage of preservation of erythropoietic and granulopoietic precursor cells in the murine bone marrow was studied using in vitro methylcellulose clonal cell culture assays and in vivo murine spleen colony assays. This study clearly demonstrates

a. Type of Spleen Colonies Induced by 6-hr Postmortem Murine Bone Marrow Cells^a

Heat exchange in the cryosurgery of Menière's disease: Experimental and clinical studies

The temperature course in the lateral semicircular canal and in the facial canal was studied in experiments during freezing of the semicircular canal. The course of the temperature was measured with thermocouples. Concurrently, the heat flow was measured, and also the total heat exchange was measured throughout the freezing period by a thermoelectric heat flowmeter incorporated in the cryotip. The measurements showed correlation between the total amount of heat exchanged, the freezing time, and the temperature in the semicircular canal. This correlation was utilized to assess and calculate (the temperature of the lateral semicircular canal) the course of the cryoprocess in vivo, where it is possible to measure the heat flow and the total heat exchange during the freezing period only.

2. Results upon Vertigo

     

4.

Integrity of lysosomes in the isolated perfused rat liver before and after exposure to dimethylsulphoxide.     

D Lee  R K Holland  A K Groufsky 《Cryobiology》1979,16(1):18-23

We have studied the integrity of lysosomes in isolated rat livers perfused for 3, 4, or 6 hr at 35 °C with BSA (40 g/l) in Krebs Ringer bicarbonate buffer. The latency and sedimentability of β-glucuronidase in homogenates of these livers was well maintained even after 6 hr. The latency and sedimentability of acid phosphatase remained at about control levels during the first 4 hr of perfusion but decreased between 4 and 6 hr. These decreases in latency and sedimentability correlated with a decrease in bile production and an increase in the rate of release of GOT into the perfusate and could indicate either intracellular disruption of lysosomes

Latencies, Sedimentabilities, and Specific Activities of Acid Phosphatase and β-Glucuronidase in Homogenates of Rat Liver Prepared before or at Various Times after Exposure to 1.4 m Me₂SO for 1 hr

	No Vertigo	Improved	Unchanged
Number of patients	7	5	3

     

5.

An experimental study using tissue culture medium as preservation and perfusion fluid     

T Nilsson  E Schueller  G P Murphy  W J Staubitz 《Cryobiology》1973,10(3):191-196

A commercially available tissue culture medium has been proven capable of preserving dog kidney function for at least 24 hr after simple cooling. The advantages of using tissue culture medium as preservation fluid instead of plasma or albumin solutions from the infectious and immunological points of view are obvious. An in vitro study was completed using the tissue

1.

Time (hr)	0	3	4	6
Acid phosphatase
Latency (%)	83.2 ± 0.8	64.7 ± 5.1	62.4 ± 7.9	68.9 ± 5.4
Sedimentability (%)	81.7 ± 0.6	77.6 ± 3.1	81.0 ± 3.4	79.4 ± 5.1
Specific activity (mIU/mg protein)	2.8 ± 0.4	2.8 ± 0.2	2.4 ± 0.4	1.6 ± 0.2
β-Glucuronidase
Latency (%)	06.8 ± 1.5	71.3 ± 4.3	74.2 ± 2.5	63.3 ± 4.5
Sedimentability (%)	69.5 ± 0.3	75.4 ± 3.2	75.0 ± 1.1	74.6 ± 2.0
Specific activity (mIU/mg protein)	1.8 ± 0.1	1.6 ± 0.1	1.6 ± 0.2	1.6 ± 0.2

     

6.

Hyperosmotic injury in mammalian cells. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures.     

S Mironescu 《Cryobiology》1978,15(2):178-191

Correlated studies on volume distributions and cation (Na⁺ and K⁺) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; $\text{w}\text{v}$). The effects superimposed by ouabain (10^?2–10^?4m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the

TABLE 2. Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension

Code (animal No.)	Perfusion time (br)	Perfusion pressure mm/Hg	Flow ml/min	Weight gain	pH	pO2 mm/Hg	Histological appearance
1	24	70-60 systolic	96	35	7.3	150–180	Grossly normal
2	24		45-40 diastolic	108	30
3	24		96	30
4	48	70-60 systolic	80	35	7.3	150–180	Grossly normal
5	48	45-40 diastolic	120	40	7.4
6	48		100	40
7	72	70-60 systolic	115	40	7.4	150–180	Slight vacuolization of the tubular cells
8	72	45-40 diastolic	96	40
9	72		80	40
10	24	70-60 systolic	110	35	7.3	150–180	Used for transplantation
11	24	45-40 diastolic	120	35
12	24		140	40
13	24		100	30
14	24		96	30

     

7.

Reproductive potential and endocrinological responses of sheep kept under controlled lighting. I. Comparative reproductive performance of four breed types of ewe     

G. Evans  T.J. Robinson 《Animal reproduction science》1980,3(1):23-37

Five ewes of each of four breed types were kept in each of two environmentally-controlled rooms over a period of 2.5 years. The daylength varied between 20 and 9 h on a 6-monthly cycle, including a period of 22 weeks during which daylength was decreased by 0.5 h per week, 2 weeks in which it increased from 9 to 20 h, and 2 weeks at 20 h; each room operated 3 months out of phase with the other. Towards the end of the period of decreasing daylength each ewe was synchronised with a progestagen sponge for 12 days, given an injection (i.m.) of 750 I.U. pregnant mares' serum gonadotrophin (PMSG) on withdrawal, and inseminated 48 and 58 h later (500 million fresh undiluted sperm per insemination). Thus, insemination occurred at a single synchronised oestrus every 6 months. Progesterone pregnancy diagnosis was performed on blood samples taken 18 days later and parturition was induced by an injection (i.m.) of 16 mg dexamethasone on day 143 of gestation. Lambs were weaned at birth, allowing the ewes approximately 5.5 weeks to recover before the next insemination and a ‘successive’ conception.The performance of the four types of ewe, starting as maidens, is tabulated.

Osmolality mOsm	Exposure (min)								Hypertonic agent
					NaCl				Sucrose
		$\text{V}{}^{\mathrm{a}}$	Na+	K+	$\text{S}{}^{\mathrm{b}}$	V	Na+	K+	S
1000	60 or less	Small	High	High	High	Normal	Low	High	High
1500–2000	60 or less	Small	High	Low	Low	Normal	Low	High	High
2000 or over	60 or more	Small or largec	High	Very low	Very low	Small	Very low	Very low	Very low

     

8.

Cell Biology: Paper alert     

《Current opinion in cell biology》2000,12(5):525-535

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.

Conceptions	Merino	Dorset Horn × Merino	Border Leic. × Merino	South Suffolk × Merino	$\text{P}$
Successive (%)	20.0	12.5	11.1	47.6	$\text{< 0.01}$
Non-successive (%)	76.0	76.9	66.7	95.0	$\text{< 0.05}$
Overall (%)	51.1	52.4	44.4	70.7	$\text{< 0.05}$
Mean litter size	1.7	1.5	1.5	2.2	$\text{< 0.05}$
Mean lambs per ewe per year	1.8	1.6	1.3	3.1	$\text{< 0.01}$

     

9.

A simple system for extending refrigerated, nonfrozen preservation of biological material using pressure.     

S E Charm  H E Longmaid  J Carver 《Cryobiology》1977,14(5):625-636

High pressure (above 238 atmg) substantially extends the refrigerated storage of highly perishable biological material in a nonfrozen state.

5. Calculated Equivalent Values of Pressure and Temperature for Reducing Reaction Rates of Horseradish Peroxidase

Contents (chosen by)
525	Cytoskeleton (Desai and Holleran)
526	Cell regulation (Roche, Servant and Weiner)
528	Nucleus and gene expression (Aasland and Weinzierl)
529	Membranes and sorting (Ponnambalam)
530	Membrane permeability (Slesinger)
531	Cell-to-cell contact and extracellular matrix (Pfaff)
533	Cell differentiation (van Roessel, Kaltschmidt, Tsang and Huckriede)
534	Cell multiplication (Sclafani)

Pressure (atmg)	Temperature (°K)	$\text{T}\text{T}{}_0$a
0	296	0.999
272	289	0.975
340	287	0.969
408	285	0.963

a

$\text{T}{}_0\text{= 296 °}\text{K}$It has been known for some time that high pressure stops microbial growth. The effect of high pressure is to reduce further the enzyme activity at refrigerated temperatures. Two enzymes studied, peroxidase and crude trypsin from red crab intestine, demonstrated this effect.A number of food materials such as fish, beef, and chicken were tested for microbial growth and organoleptic qualities after high-pressure storage in a simple 14-liter pressure chamber. Pressure was generated by a hand pump. The results indicated that after 30 days those items held in a non-frozen state at ?3 °C and 238 atmg were not significantly different microbiologically and organoleptically from frozen controls at atmospheric pressure and ?20 °C.This system should be useful for the preservation of biological materials where freezing or thawing effects are undesirable or unknown.The energy saved compared to freezing should also be considered. Only 62% of the energy is required for storage at ?3 °C as compared with frozen storage at ?20 °C, and about 28 cal/g must be removed in cooling to ?3 °C as compared with 120 cal/g in cooling to ?20 °C.

     

Integrity of lysosomes in the isolated perfused rat liver before and after exposure to dimethylsulphoxide.

We have studied the integrity of lysosomes in isolated rat livers perfused for 3, 4, or 6 hr at 35 °C with BSA (40 g/l) in Krebs Ringer bicarbonate buffer. The latency and sedimentability of β-glucuronidase in homogenates of these livers was well maintained even after 6 hr. The latency and sedimentability of acid phosphatase remained at about control levels during the first 4 hr of perfusion but decreased between 4 and 6 hr. These decreases in latency and sedimentability correlated with a decrease in bile production and an increase in the rate of release of GOT into the perfusate and could indicate either intracellular disruption of lysosomes

Latencies, Sedimentabilities, and Specific Activities of Acid Phosphatase and β-Glucuronidase in Homogenates of Rat Liver Prepared before or at Various Times after Exposure to 1.4 m Me₂SO for 1 hr

An experimental study using tissue culture medium as preservation and perfusion fluid

A commercially available tissue culture medium has been proven capable of preserving dog kidney function for at least 24 hr after simple cooling. The advantages of using tissue culture medium as preservation fluid instead of plasma or albumin solutions from the infectious and immunological points of view are obvious. An in vitro study was completed using the tissue

1.

Hyperosmotic injury in mammalian cells. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures.

Correlated studies on volume distributions and cation (Na⁺ and K⁺) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; $\text{w}\text{v}$). The effects superimposed by ouabain (10^?2–10^?4m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the

TABLE 2. Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension

Reproductive potential and endocrinological responses of sheep kept under controlled lighting. I. Comparative reproductive performance of four breed types of ewe

Five ewes of each of four breed types were kept in each of two environmentally-controlled rooms over a period of 2.5 years. The daylength varied between 20 and 9 h on a 6-monthly cycle, including a period of 22 weeks during which daylength was decreased by 0.5 h per week, 2 weeks in which it increased from 9 to 20 h, and 2 weeks at 20 h; each room operated 3 months out of phase with the other. Towards the end of the period of decreasing daylength each ewe was synchronised with a progestagen sponge for 12 days, given an injection (i.m.) of 750 I.U. pregnant mares' serum gonadotrophin (PMSG) on withdrawal, and inseminated 48 and 58 h later (500 million fresh undiluted sperm per insemination). Thus, insemination occurred at a single synchronised oestrus every 6 months. Progesterone pregnancy diagnosis was performed on blood samples taken 18 days later and parturition was induced by an injection (i.m.) of 16 mg dexamethasone on day 143 of gestation. Lambs were weaned at birth, allowing the ewes approximately 5.5 weeks to recover before the next insemination and a ‘successive’ conception.The performance of the four types of ewe, starting as maidens, is tabulated.

Cell Biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.

A simple system for extending refrigerated, nonfrozen preservation of biological material using pressure.

High pressure (above 238 atmg) substantially extends the refrigerated storage of highly perishable biological material in a nonfrozen state.

5. Calculated Equivalent Values of Pressure and Temperature for Reducing Reaction Rates of Horseradish Peroxidase

Clinical challenges

Glaucomatocyclitic crisis should be considered in the differential diagnosis of unilaterally increased IOP. A careful history, slit-lamp examination and gonioscopic examination will assist the examiner in making an accurate diagnosis.The etiology of this syndrome remains unknown. If indeed it is a disturbance of the anterior intraocular vasculature secondary to an autonomic abnormality, any of the proposed etiologies might possibly trigger an attack (Table 2).Treatment at this time should consist of 1 gH. 0.5% apraclonidine given in office or a topical steroid, cycloplegic, and ocular hypotensive agent combination. Follow-up should be within 24 h to monitor for IOP reduction. If treatment with apraclonidine is initiated, additional drops should be instilled as needed to lower the IOP to acceptable levels. Prophylactic treatment with topical steroids has been considered in this mostly benign syndrome [14]. However, the potential side-effects of long-term steroid use and the unpredictable frequency of glaucomatocyclitic crises may preclude steroid use in these cases. Table 2. Proposed etiological factors and diseases related to glaucomatocyclitis crisis.      

11.

Calorimetric determination of base-stacking enthalpies in double-helical DNA molecules     

Luis A. Marky  Kenneth J. Breslauer 《Biopolymers》1982,21(11):2185-2194

Differential scanning calorimetry was used to directly determine the transition enthalpies accompanying the duplex-to-single-strand transition of poly[d(AT)], poly(dA)·poly(dT), poly[d(AC)]·poly[d(TG)], and d(GCGCGC). The calorimetric data allow us to define the following average base-stacking enthalpies:

Calorimetric determination of base-stacking enthalpies in double-helical DNA molecules

Differential scanning calorimetry was used to directly determine the transition enthalpies accompanying the duplex-to-single-strand transition of poly[d(AT)], poly(dA)·poly(dT), poly[d(AC)]·poly[d(TG)], and d(GCGCGC). The calorimetric data allow us to define the following average base-stacking enthalpies:

Comparison of the Efficacy of the Two Tetracycline‐Containing Sequential Therapy Regimens for the Eradication of Helicobacter Pylori: 5 days Versus 14 days Amoxicillin

Aim: To compare the efficacy of 14‐day and 5‐day amoxicillin treatment on the eradication rate during tetracycline containing sequential H. pylori therapy, and also to compare the eradication rate of this regimen with those used in similar studies performed in Turkey. Method: This study included 112 patients infected with H. pylori that were randomized into 2 groups. In group A, patients (n = 56) received pantoprazole (40 mg BID) and amoxicillin (1 g BID) for 5 days, followed by pantoprazole (40 mg BID), tetracycline (500 mg QID), and metronidazole (500 mg TID) for the remaining 9 days. In group B, patients (n = 56) received pantoprazole (40 mg BID) and amoxicillin (1 g BID) for 5 days, followed by pantoprazole (40 mg BID), tetracycline (500 mg QID), metronidazole (500 mg TID), and amoxicillin (1 g BID) for the remaining 9 days. Eradication rates were calculated using both intention‐to‐treat (ITT) and per‐protocol (PP) analyses. Results: In all, 112 patients were subjected to ITT analysis and 109 patients completed the study. In group A, H. pylori eradication was achieved in 46 (82.1%) of the 56 patients included in the ITT analysis and in 46 (83.6%) of the 55 patients included in the PP analysis. In group B, H. pylori eradication was achieved in 44 (78.57%) of the 56 patients included in the ITT analysis and in 44 (81.48%) of the 54 patients included in the PP analysis ( Table 2 ). The eradication rates were not statistically significant between the 2 groups (p > .005).

Table 2. Eradication rates in the two study groups

Archaeology of Eukaryotic DNA Replication

Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously uncharacterized archaeal proteins involved in replication and currently reveal a nearly complete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were comparable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionalization of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific duplications of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal replication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages.Double-stranded DNA is the molecule that carries genetic information in all cellular life-forms; thus, replication of this genetic material is a fundamental physiological process that requires high accuracy and efficiency (Kornberg and Baker 2005). The general mechanism and principles of DNA replication are common in all three domains of life—archaea, bacteria, and eukaryotes—and include recognition of defined origins, melting DNA with the aid of dedicated helicases, RNA priming by the dedicated primase, recruitment of DNA polymerases and processivity factors, replication fork formation, and simultaneous replication of leading and lagging strands, the latter via Okazaki fragments (Kornberg and Baker 2005; Barry and Bell 2006; Hamdan and Richardson 2009; Hamdan and van Oijen 2010). Thus, it was a major surprise when it became clear that the protein machineries responsible for this complex process are drastically different, especially in bacteria compared with archaea and eukarya. The core components of the bacterial replication systems, such as DNA polymerase, primase, and replication helicase, are unrelated or only distantly related to their counterparts in the archaeal/eukaryotic replication apparatus (Edgell 1997; Leipe et al. 1999).The existence of two distinct molecular machines for genome replication has raised obvious questions on the nature of the replication system in the last universal common ancestor (LUCA) of all extant cellular life-forms, and three groups of hypotheses have been proposed (Leipe et al. 1999; Forterre 2002; Koonin 2005, 2006, 2009; Glansdorff 2008; McGeoch and Bell 2008): (1) The replication systems in Bacteria and in the archaeo–eukaryotic lineage originated independently from an RNA-genome LUCA or from a noncellular ancestral state that encompassed a mix of genetic elements with diverse replication strategies and molecular machineries. (2) The LUCA was a typical cellular life-form that possessed either the archaeal or the bacterial replication apparatus in which several key components have been replaced in the other major cellular lineage. (3) The LUCA was a complex cellular life-form that possessed both replication systems, so that the differentiation of the bacterial and the archaeo–eukaryotic replication machineries occurred as a result of genome streamlining in both lines of descent that was accompanied by differential loss of components. With regard to the possible substitution of replication systems, a plausible mechanism could be replicon takeover (Forterre 2006; McGeoch and Bell 2008). Under the replicon takeover hypothesis, mobile elements introduce into cells a new replication system or its components, which can displace the original replication system through one or several instances of integration of the given element into the host genome accompanied by inactivation of the host replication genes and/or origins of replication. This scenario is compatible with the experimental results showing that DNA replication DNA in Escherichia coli with an inactivated DnaAgene or origin of replication can be rescued by the replication apparatus of R1 or F1 plasmids integrated into the bacterial chromosome (Bernander et al. 1991; Koppes 1992). Furthermore, genome analysis suggests frequent replicon fusion in archaea and bacteria (McGeoch and Bell 2008); in particular, such events are implied by the observation that in archaeal genomes, genes encoding multiple paralogs of the replication helicase MCM and origins of replication are associated with mobile elements (Robinson and Bell 2007; Krupovic et al. 2010). Replicon fusion also is a plausible path from a single origin of replication that is typical of bacteria to multiple origins present in archaea and eukaryotes. However, all the evidence in support of frequent replicon fusion and the plausibility of replicon takeover notwithstanding, there is no evidence of displacement of the bacterial replication apparatus with the archaeal version introduced by mobile elements, or vice versa, displacement of the archaeal machinery with the bacterial version, despite the rapid accumulation of diverse bacterial and archaeal genome sequences. Thus, the displacement scenarios of DNA replication machinery evolution are so far not supported by comparative genomic data.Regardless of the nature of the DNA replication system (if any) in the LUCA and the underlying causes of the archaeo–bacterial dichotomy of replication machineries, the similarity between the archaeal and eukaryotic replication systems is striking (Leipe et al. 1999; Bell and Dutta 2002; Bohlke et al. 2002; Kelman and White 2005; Barry and Bell 2006). Thus, the archaeal replication system appears to be an ancestral version of the eukaryotic system and hence a good model for functional and structural studies aimed at gaining mechanistic insights into eukaryotic replication.

Table 1.

The relationship between archaeal and eukaryotic replication systems

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol γ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase γ (pol γ)³ bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol γ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3′ → 5′ exonuclease, and 5′-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol γ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol γ, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and recessive forms of progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, ataxia-neuropathy syndromes, and male infertility (tools.niehs.nih.gov/polg) (9).Alpers syndrome, a hepatocerebral mtDNA depletion disorder, and myocerebrohepatopathy are rare heritable autosomal recessive diseases primarily affecting young children (10–12). These diseases generally manifest during the first few weeks to years of life, and symptoms gradually develop in a stepwise manner eventually leading to death. Alpers syndrome is characterized by refractory seizures, psychomotor regression, and hepatic failure (11, 12). Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16, 17).During the initial cloning and sequencing of the human, Drosophila, and chicken pol γ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol γ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19–21). A high concentration of disease mutations, many associated with Alpers syndrome, is found in the thumb subdomain.Here we investigated six mitochondrial disease mutations clustered in the N-terminal portion of the polymerase domain of the enzyme (Fig. 1A). Four mutations (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) reside in the thumb subdomain and two (Q879H, c.2637g→t and T885S, c.2653a→t) are located in the palm subdomain. These mutations are associated with Alpers, PEO, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), ataxia-neuropathy syndrome, Leigh syndrome, and myocerebrohepatopathy (