A cladistic analysis of apolipoprotein B gene variation and plasma lipid and apolipoprotein B levels, using familial data

Any functionally important mutation is embedded in an evolutionary matrix of other mutations. Cladistic analysis, based on this, is a method of investigating gene effects using a haplotype phylogeny to define a set of tests which localize causal mutations to branches of the phylogeny. Previous implementations of cladistic analysis have not addressed the issue of analyzing data from related individuals, though in human studies, family data are usually needed to obtain unambiguous haplotypes. In this study, a method of cladistic analysis is described in which haplotype effects are parameterized in a linear model which accounts for familial correlations. The method was used to study the effect of apolipoprotein (Apo) B gene variation on total-, LDL-, and HDL-cholesterol, triglyceride, and Apo B levels in 121 French families. Five polymorphisms defined Apo B haplotypes: the signal peptide Insertion/deletion, Bsp 1286I, XbaI, MspI, and EcoRI. Eleven haplotypes were found, and a haplotype phylogeny was constructed and used to define a set of tests of haplotype effects on lipid and apo B levels.^ This new method of cladistic analysis, the parametric method, found significant effects for single haplotypes for all variables. For HDL-cholesterol, 3 clusters of evolutionarily-related haplotypes affecting levels were found. Haplotype effects accounted for about 10% of the genetic variance of triglyceride and HDL-cholesterol levels. The results of the parametric method were compared to those of a method of cladistic analysis based on permutational testing. The permutational method detected fewer haplotype effects, even when modified to account for correlations within families. Simulation studies exploring these differences found evidence of systematic errors in the permutational method due to the process by which haplotype groups were selected for testing.^ The applicability of cladistic analysis to human data was shown. The parametric method is suggested as an improvement over the permutational method. This study has identified candidate haplotypes for sequence comparisons in order to locate the functional mutations in the Apo B gene which may influence plasma lipid levels. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean Grey cattle.

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.

A 19-Mb de novo deletion on BTA 22 including MITF leads to microphthalmia and the absence of pigmentation in a Holstein calf.

Mutations in MITF lead to a large variety of phenotypes in human, mice and other species. They mostly affect pigmentation and hearing, whereas in mice, they may additionally cause microphthalmia and osteopetrosis. In this study, we report a single case of a Holstein calf with lack of pigmentation and microphthalmia born to healthy parents. Mendelian analysis of high-density SNP genotypes reveals a large number of parentage errors showing missing paternal alleles in the offspring, indicating a deletion encompassing 19 Mb on BTA 22. The genomic deletion affects the paternal allele and includes MITF and 131 other annotated genes. As the calf shows only one copy of the BTA 22 segment, the observed phenotype is probably caused by haploinsufficiency of the genes in that genomic region. Both the observed lack of skin pigmentation and reduced eye size can most likely be explained by a lack of MITF function.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

A Deletion in the VLDLR Gene in Eurasier Dogs with Cerebellar Hypoplasia Resembling a Dandy-Walker-Like Malformation (DWLM).

Dandy-Walker-like malformation (DWLM) is the result of aberrant brain development and mainly characterized by cerebellar hypoplasia. DWLM affected dogs display a non-progressive cerebellar ataxia. Several DWLM cases were recently observed in the Eurasier dog breed, which strongly suggested a monogenic autosomal recessive inheritance in this breed. We performed a genome-wide association study (GWAS) with 9 cases and 11 controls and found the best association of DWLM with markers on chromosome 1. Subsequent homozygosity mapping confirmed that all 9 cases were homozygous for a shared haplotype in this region, which delineated a critical interval of 3.35 Mb. We sequenced the genome of an affected Eurasier and compared it with the Boxer reference genome and 47 control genomes of dogs from other breeds. This analysis revealed 4 private non-synonymous variants in the critical interval of the affected Eurasier. We genotyped these variants in additional dogs and found perfect association for only one of these variants, a single base deletion in the VLDLR gene encoding the very low density lipoprotein receptor. This variant, VLDLR:c.1713delC is predicted to cause a frameshift and premature stop codon (p.W572Gfs*10). Variants in the VLDLR gene have been shown to cause congenital cerebellar ataxia and mental retardation in human patients and Vldlr knockout mice also display an ataxia phenotype. Our combined genetic data together with the functional knowledge on the VLDLR gene from other species thus strongly suggest that VLDLR:c.1713delC is indeed causing DWLM in Eurasier dogs.

Congenital aural atresia associated with agenesis of internal carotid artery in a girl with a FOXI3 deletion.

We report on the molecular characterization of a microdeletion of approximately 2.5 Mb at 2p11.2 in a female baby with left congenital aural atresia, microtia, and ipsilateral internal carotid artery agenesis. The deletion was characterized by fluorescence in situ hybridization, array comparative genomic hybridization, and whole genome re-sequencing. Among the genes present in the deleted region, we focused our attention on the FOXI3 gene. Foxi3 is a member of the Foxi class of Forkhead transcription factors. In mouse, chicken and zebrafish Foxi3 homologues are expressed in the ectoderm and endoderm giving rise to elements of the jaw as well as external, middle and inner ear. Homozygous Foxi3-/- mice have recently been generated and show a complete absence of the inner, middle, and external ears as well as severe defects in the jaw and palate. Recently, a 7-bp duplication within exon 1 of FOXI3 that produces a frameshift and a premature stop codon was found in hairless dogs. Mild malformations of the outer auditory canal (closed ear canal) and ear lobe have also been noted in a fraction of FOXI3 heterozygote Peruvian hairless dogs. Based on the phenotypes of Foxi3 mutant animals, we propose that FOXI3 may be responsible for the phenotypic features of our patient. Further characterization of the genomic region and the analysis of similar patients may help to demonstrate this point. © 2015 Wiley Periodicals, Inc.

Epidermolysis bullosa in Danish Hereford calves is caused by a deletion in LAMC2 gene.

BACKGROUND Heritable forms of epidermolysis bullosa (EB) constitute a heterogeneous group of skin disorders of genetic aetiology that are characterised by skin and mucous membrane blistering and ulceration in response to even minor trauma. Here we report the occurrence of EB in three Danish Hereford cattle from one herd. RESULTS Two of the animals were necropsied and showed oral mucosal blistering, skin ulcerations and partly loss of horn on the claws. Lesions were histologically characterized by subepidermal blisters and ulcers. Analysis of the family tree indicated that inbreeding and the transmission of a single recessive mutation from a common ancestor could be causative. We performed whole genome sequencing of one affected calf and searched all coding DNA variants. Thereby, we detected a homozygous 2.4 kb deletion encompassing the first exon of the LAMC2 gene, encoding for laminin gamma 2 protein. This loss of function mutation completely removes the start codon of this gene and is therefore predicted to be completely disruptive. The deletion co-segregates with the EB phenotype in the family and absent in normal cattle of various breeds. Verifying the homozygous private variants present in candidate genes allowed us to quickly identify the causative mutation and contribute to the final diagnosis of junctional EB in Hereford cattle. CONCLUSIONS Our investigation confirms the known role of laminin gamma 2 in EB aetiology and shows the importance of whole genome sequencing in the analysis of rare diseases in livestock.

DNA-based diagnosis of rare diseases in veterinary medicine: a 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in Charolais cattle.

BACKGROUND Rare diseases in livestock animals are traditionally poorly diagnosed. Other than clinical description and pathological examination, the underlying causes have, for the most part, remained unknown. A single case of congenital skin fragility in cattle was observed, necropsy, histological and ultrastructural examinations were carried out and whole genome sequencing was utilized to identify the causative mutation. RESULTS A single purebred female Charolais calf with severe skin lesions was delivered full-term and died spontaneously after birth. The clinical and pathological findings exactly matched the gross description given by previous reports on epitheliogenesis imperfecta and epidermolysis bullosa (EB) in cattle. Histological and ultrastructural changes were consistent with EB junctionalis (EBJ). Genetic analysis revealed a previously unpublished ITGB4 loss-of-function mutation; the affected calf was homozygous for a 4.4 kb deletion involving exons 17 to 22, and the dam carried a single copy of the deletion indicating recessive inheritance. The homozygous mutant genotype did not occur in healthy controls of various breeds but some heterozygous carriers were found among Charolais cattle belonging to the affected herd. The mutant allele was absent in a representative sample of unrelated sires of the German Charolais population. CONCLUSION This is the first time in which a recessively inherited ITGB4 associated EBJ has been reported in cattle. The identification of heterozygous carriers is of importance in avoiding the transmission of this defect in future. Current DNA sequencing methods offer a powerful tool for understanding the genetic background of rare diseases in domestic animals having a reference genome sequence available.

Post-colonial (American?) English Presence on the North-Western Pacific Island of Kosrae: A cross-linguistic analysis of the overlaps and divergences of Kosraean English compared to standard American English

Kosrae is the most remote island of the Federated States of Micronesia (FSM), with a population of less than 7,000 inhabitants, located in the Pacific Ocean between Hawaii and Guam. FSM is an independent sovereign nation consisting of four states in total: Pohnpei, Chuuk, Yap, and Kosrae. Having passed through the hands of Spain, Germany and Japan, the United States gained administrative control of FSM after WWII, as commissioned by the UN. The FSM became an independent nation in 1986 while still retaining affiliation with the US under a ‘Compact of Free Association’. Now both Kosraean and English are considered to be the two official languages and the variety of Kosraean English which has arisen proves for an interesting comparative study. In order to obtain the relevant data, I spent three months on the island of Kosrae, interviewing 90 local speakers, ranging in age (16-70), occupation, sex and time spent off island. The 45 minute long interviews were informal but supported by participant information to capture relevant data and conversations were guided in a way that aimed to reveal language and cultural attitudes. With reference to these samples, I examine the effects of American English on the language use in Kosrae. This paper aims to present a broad analysis of phonological, morphosyntactic and pragmatic features, such as pro-dropping, discourse markers and other practices in order to demonstrate the similarities and differences between the two varieties, which are coming to shape the variety developing on Kosrae. Having transcribed conversations using the tool Elan, I will put particular focus on [h] deletion and insertion, a rare occurrence found in a variety of post-colonial American English which I believe is of particular interest. I assess the presence of English in Kosrae with reference to sociological influences, past and present. First, I discuss the extralinguistic factors which have shaped the English that is currently used on Kosrae, including migration between US and FSM, and English as a language of administration, social media usage and visual media presence. Secondly, I assess the use of English in this community in light of Schneider’s (2007) ‘Dynamic Model’, with reference to America’s contribution as an ‘exploitation colony’ as defined by Mufwene (2001). Finally, an overview of the salient linguistic characteristics of Kosraean English, based on the data collected will be presented and compared to features associated with standard American English in view of examining overlap and divergence. The overall objective is to present a cross-linguistic description of a hitherto unexamined English emerging in a postcolonial environment with a juxtaposed contact variety. Mufwene, Salikoko S. 2001. The ecology of language evolution. Cambridge: Cambridge University Press. Schneider, E. (2007). Postcolonial Englishes. Cambridge: Cambridge University Press. Segal, H.G. (1989) Kosrae, The Sleeping Lady Awakens. Kosrae: Kosrae Tourist Division, Dept. Of Conservation and Development. Keywords: American English, Global English, Pacific English, Morphosyntactic, Phonological, Variation, Discourse

A variationist approach to the examination of insertion and deletion of /h/ in Kosraean English

I report on language variation in the unresearched variety of English emerging on Kosrae, Federated States of Micronesia. English is spoken as the inter-island lingua franca throughout Micronesia and has been the official language of FSM since gaining its independence in 1986, though still retaining close ties with the US through and economic “compact” agreement. I present here an analysis of a corpus of over 90 Kosraean English speakers, compiled during a three month fieldwork trip to the island in the Western Pacific. The 45 minute sociolinguistically sensitive recordings are drawn from a corpus of old and young, with varying levels of education and occupations, and off-island experiences. In the paper I analyse two variables. The first variable is the realisation of /h/, often subject to deletion in both L1 and L2 varieties of English. Such occurrences are commonly associated with Cockney English, but also found in Caribbean English and the postcolonial English of Australia. For example:  Male, 31: yeah I build their house their local huts and they pay me /h/ deletion is frequent in Kosraean English, but, perhaps expectedly, occurs slightly less among people with higher contact with American English, through having spent longer periods off island. The second feature under scrutiny is the variable epenthesis of [h] to provide a consonantal onset to vowel-initial syllables.  Male, 31: that guy is really hold now This practice is also found beyond Kosraean English. Previous studies find h-epenthesis arising in L1 varieties including Newfoundland and Tristan de Cunha English, while similar manifestations are identified in Francophone L2 learners of English. My variationist statistical analysis has shown [h] insertion:  to disproportionately occur intervocalically;  to be constrained by both speaker gender and age: older males are much more likely to epenthesis [h] in their speech;  to be more likely in the onset of stressed as opposed to unstressed syllables. In light of the findings of my analysis, I consider the relationship between h-deletion and h-epenthesis, the plausibility of hypercorrection as a motivation for the variation, and the potential influence of the substrate language, alongside sociolinguistic factors such as attitudes towards the US based on mobility. The analysis sheds light on the extent to which different varieties share this characteristic and the comparability of them in terms of linguistic constraints and attributes. Clarke, S. (2010). Newfoundland and Labrador English. Edinburgh: Edinburgh University Press Hackert, S. (2004). Urban Bahamian Creole: System and Variation. Varieties of English Around the World G32. Amsterdam: Benjamins Milroy, J. (1983). On the Sociolinguistic History of H-dropping in English in Current topics in English historical linguistics: Odense UP

Phylogenetic analysis of receptor FgfrL1 shows divergence of the C-terminal end in rodents

FGFRL1 is a member of the fibroblast growth factor receptor (FGFR) family. Similar to the classical receptors FGFR1-FGFR4, it contains three extracellular Ig-like domains and a single transmembrane domain. However, it lacks the intracellular tyrosine kinase domain that would be required for signal transduction, but instead contains a short intracellular tail with a peculiar histidine-rich motif. This motif has been conserved during evolution from mollusks to echinoderms and vertebrates. Only the sequences of FgfrL1 from a few rodents diverge at the C-terminal region from the canonical sequence, as they appear to have suffered a frameshift mutation within the histidine-rich motif. This mutation is observed in mouse, rat and hamster, but not in the closely related rodents mole rat (Nannospalax) and jerboa (Jaculus), suggesting that it has occurred after branching of the Muridae and Cricetidae from the Dipodidae and Spalacidae. The consequence of the frameshift is a deletion of a few histidine residues and an extension of the C-terminus by about 40 unrelated amino acids. A similar frameshift mutation has also been observed in a human patient with a craniosynostosis syndrome as well as in several patients with colorectal cancer and bladder tumors, suggesting that the histidine-rich motif is prone to mutation. The reason why this motif was conserved during evolution in most species, but not in mice, is not clear.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

An analysis of the function of region 1.2 of the primary sigma factor, sigma 70, from Escherichia coli

The sigma (σ) subunit of eubacterial RNA polymerase is required for recognition of and transcription initiation from promoter DNA sequences. One family of sigma factors includes those related to the primary sigma factor from E. coli, σ70. Members of the σ70 family have four highly conserved domains, of which regions 2 through 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by σ 70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter specific binding by holoenzyme. This study primarily investigates the functional role of region 1.2 during transcription initiation. In vivo and in vitro characterization of thirty-two single amino acid substitutions targeted to region 1.2 of E. coli σ70 as well as a deletion of region 1.2, revealed that mutations in region 1.2 can affect promoter binding, open complex formation, initiated complex formation, and the transition from abortive transcription to elongation. The relative degree of solvent exposure of several positions in region 1.2 has been determined, with positions 116 and 122 likely to be located near the surface of σ70. ^ During the course of this study, the existence of two “wild type” variants of E. coli σ70 was discovered. The identity of amino acid 149 has been reported variably as either arginine or aspartic acid in published articles and in online databases. In vivo and in vitro characterization of the two reported variations of E. coli σ70 (N149 and D149) has determined that the two variants are functionally equivalent. However, in vivo and in vitro characterization of single amino acid substitutions and a region 1.2 deletion in the context of each variant background revealed that the behavior of some mutations are greatly affected by the identity of amino acid 149. ^